Post-translational modifications change function of most proteins. The ubiquitin system gives rise to the regulation of almost every cellular activity through proteolytic and non-proteolytic events, including protein degradation by the lysosomal pathway or through the 26S proteasome and autophagy, protein in every cellular activity the activation, and changed localization (1).
This review focuses on an up-to-date understanding of the functions of E3 ligases in cancers and debates the perspectives of cancer cells that rely on inhibition or activation of ubiquitylation of target proteins. The covalent attachment of ubiquitin to a target protein is catalyzed by the sequential action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). The E3 ubiquitination step determines the overall specificity for ubiquitination of the target protein, and the activated ubiquitin is usually transferred to a lysine residue in the substrate. Ubiquitination of proteins by a small 76 amino acid protein plays an essential role in various aspects of the DNA damage response (DDR) (2). In the case of ubiquitination signaling, the mechanism of cancer differentiation was insufficient due to the complication of ubiquitin signaling and because many aspects of the essential biology of this signaling are not yet completely understood (1). E3 ligases consist of a large family of more than 600 proteins (3), and a few E3 ligases have been classified based on their conserved structural domains (e.g., RING, RING-SCF, or HECT domains) (4, 5).
Ubiquitination of double-strand break (DSB) repair genes and proteins plays an essential role in cancer because their translocation, interaction, alteration, and regulation can affect the DNA repair pathway, making cancer cells more resistant or more prone to genomic instability. The DDR-associated E3 ligases modify histones and DDR-related proteins (Fig. 1). The failure of DNA repair results in genetic instability, and thus the detailed E3 ligase mechanism plays an important role in cancer cell instability. Therefore, it is necessary to approach effective treatment methods through mechanism research.
The scheme of DSBs following IR (Ionizing Radiation) or NCS (Neocarzinostatin) treatment activates the Mre11/Rad50/Nbs1-Ataxia telangiectasia-mutated (MRN/ATM) complex and several E3 ligases at DSBs. In addition to induction of the H2A/γH2AX-dependent signaling, which results in the recruitment of MDC1, RNF8, RNF168, BRCA1, and 53BP1 to DSBs. DDR-related E3 ligase genetic syndromes can also lead to premature aging or cancer. In this scheme, we will describe cancer associated with DDR-related E3 ligases.
E3 ligase has an essential role in recruiting key DNA DSB repair factors to DNA damage sites. E3 ligases add one or multiple ubiquitin chains at one or several lysine residues, such as K6, K11, K27, K29, K33, K48, and K63. In most cases, K48-linked and K11-linked poly-ubiquitin chains are signals for degradation by the proteasome. Conversely, K63-linked poly-ubiquitin chains cause a change in the substrate activity, substrate signaling, and localization of the substrate (6). Activation of the MRN complex promotes initiation of the DDR and ATM recruitment to DSBs. SKP2 affects MRN complex-mediated ATM activation by triggering K63-linked NBS1 at DSB sites. Phosphorylated FBXW7 by ATM promotes K63-linked poly-ubiquitination of XRCC4 to facilitate recruitment of Ku70/80 for nonhomologous end joining (NHEJ) repair (7-9). WWP2 induces DNA-PKcs (Protein Kinase, Catalytic Subunit)-dependent proteasomal degradation of RNAPII. RNAPII inhibits DNA-PK, XRCC4, DNA ligase IV (LigIV), and XLF (10). RNAPII degradation protects NHEJ machinery in DSB repair sites.
Ubiquitination of histones H2A, H2B, H3, and H4 is important for DNA repair signaling. Subsequent to DSBs, γH2A, K13, and K15 are ubiquitinated by RNF168, and K127 and K129 of BRCA1/BRCA1-associated RING domain protein 1 (BARD1) are ubiquitinated (11). The DNA DSB of MDC1 is polyubiquitinated at K63 by RNF8 and subsequently recognized by RNF168, leading to the recruitment of repair proteins, such as BRCA1 and 53BP1 (12). HERC2 recruited by MDC1 promotes RNF8 oligomerization and expedites the formation of the HERC2/MDC1/RNF8 complex, following RNF8-mediated recruitment of RNF168 (13). The dimethylation of H4K20 is recruitment of 53BP1, where it commends repair pathway selection decisions by limiting DNA end resection at DSB sites. Accumulation of 53BP1 disrupt DNA end resection and homologous recombination (HR) whereas implement NHEJ (14). The K127/129 of H2A binds to BRCA1/BARD1 and promotes end resection and HR (15). The PALB2/BRCA1 complex recruits RAD51 and BRCA2 and promotes RAD51 replacement of RPA (16). Although HR is usually valuable for preserve genome integrity, DSB repair by HR is highly inhibited in G1 cells in non-dividing cells. KEAP1 inhibits interaction of PALB2/BRCA1 and dissociation of BRCA1 inhibits HR in G1 phase (17). The ubiquitination of RAP80 binds BRCA1 complex to DSB sites. The RAP80/BRCA1 complex is required for efficient BRCA1 recruitment, that suppresses excessive DSB end processing, HR repair, and chromosomal instability. BRCA1 induces ubiquitination of CtIP and promotes its chromatin-loading at the G2/M checkpoint (18). The interaction of BRCA1/CtIP was initially implicated in the G2/M checkpoint and apoptosis regulation. The relation of BRCA1/CtIP facilitates HR through ubiquitination. However, separation of the BRCA1/CtIP complex in the G2/M checkpoint accelerates apoptosis in damaged cells. RNF138 recruits MRE11 but not CtIP at ssDNA overhangs resembling initial processing of DSBs RNF138 mediates Ku80 ubiquitylation following DSB repair pathway utilization, Ku70/80, and MRN and promotes RPA loading, CtIP, and exonuclease 1 (EXO1), and hence, facilitates HR (19). RNF168, competing with RNF169, is recruited to DSBs, eliminates RAP80 by histone, and facilitates HR. RNF20/RNF40 mono-ubiquitinates histone H2BK120 at DSBs, which leads to chromatin relaxation. RNF126 upregulates HR through BRCA1 interacting with E2F1 (20). RNF126 promotes K48-linked ubiquitination of Ku70/Ku80, loosening from DNA DSB sites, which recruits proteins that mediate repair by NHEJ (21). Finally, RNF126 directly ubiquitinates RNF168, thereby inhibiting RNF168-mediated ubiquitination of γH2AX (22).
Several cancers are caused by endogenous and exogenous DNA damage. Defects in the DNA DSB mechanisms are triggered by chromosomal abnormalities that cause tumorigenesis and genomic instability. DNA DSB factors and ubiquitination of histones play crucial functions in recruiting proteins to damaged DSB sites and the repair operation (23). In this review, we study the DDR-related E3 ligase genes, such as RNF8, RNF20/40, RNF53 (BRCA1), RNF126, RNF138, RNF168, RNF169, SKP2, FBXW7, HERC2, and WWP2 (Fig. 2).
Once DSB occurs, auto-activated ATM rapidly phosphorylates γH2AX at S139, recruiting MDC1 to the DSB sites (24-26). γH2AX recruits the E3 ligases RNF8 and RNF168 (27). RNF8 and RNF168 promote the K63-linked polyubiquitination of γH2AX on K13 and K15. As a result, 53BP1 and BRCA1 are competitively combined polyubiquitination of γH2AX to determine repair pathway (28). Furthermore, RNF8-mediated ubiquitination of NBS1 K435 is essential for HR. The genetic mutations and expression of RNF8 were investigated using the cBioPortal for Cancer Genomics (cBioPortal) databases. The results found that across 584 ovarian cancers in the cBioPortal, RNF8 exhibited high gene alteration frequencies (4.97%, respectively), and RNF8 gene amplification was very high. The rates of point mutations in RNF8 were 3.97% (21 cases) in 529 uterine corpus endometrial carcinomas. These datasets showed significantly increased expression and mutation of RNF8 in cancers, such as ovarian serous cystadenocarcinoma, uterine corpus endometrial carcinoma, and skin cutaneous melanoma. MiR-214 inhibits RNF8, thereby disrupting DNA repair to induce chromosomal instability in ovarian cancers (29). According to a recent study, RNF8 causes epithelial-mesenchymal transition (EMT) and CSC self-renewal in triple-negative breast cancer cells. Furthermore, RNF8 triggers cancer cell invasion and migration and cancer metastasis, with the result that cancer cells induce chemoresistance and tumor aggressiveness (30).
Numerous histone-modifying E3 ligases have been implicated in DSBs. RNF20/RNF40 mono-ubiquitinylates histone H2B on K120 (H2Bub). It is required for DSB repair leading to HR and class switch recombination, and also induces the recruitment of BRCA1 and RAD51 (31, 32). RNF20/40-dependent H2Bub is essential for transcription of H3K4me3 and H3K79me2. The RNF20/RNF40 association with the RNA polymerase II-associated factor 1 (PAF1) complex regulates H2Bub at the coding regions of transcriptionally active downstream genes (33-35). The mutation of RNF20 (>5% of samples) has been detected in three tumor types: uterine corpus endometrial, skin cutaneous melanoma carcinoma, and colorectal adenocarcinoma. Despite that, the prognostic significance of RNF20 mutations in uterine carcinosarcoma remains uncertain. P53 regulates the transcription of p21 and PUMA through the RNF20/RNF40 complex-dependent recruitment of H2Bub. A lack of the RNF20/RNF40 complex inhibited not only H2Bub but also the mature mRNA of p21 and PUMA. PUMA regulates mRNA splicing for tumor suppressor genes in colon cancer (36).
The breast and ovarian cancer gene, BRCA1, encodes a RING domain flanked by long α-helices. The BRCA1/BARD1 heterodimer has an essential role in E3 ligase activity and participates in chromosome stability and cell proliferation (37). BRCA1/BARD1 ubiquitylates K125/127/129 of H2A (38). SMARCAD1 interacts ubiquitinated H2A. SMARCAD1 is required for 53BP1 repositioning (15). The ubiquitination signaling of γH2AX results in the ultimate recruitment of DSB repair proteins 53BP1 and BRCA1. 53BP1 is associated with cNHEJ repair of the DSB, while BRCA1 regulates HR-mediated repair. The overall incidence of DDR pathway alterations of BRCA1 in the provisional datasets of The Cancer Genome Atlas (TCGA) database was 8.88% of the 529 samples. There were 47 mutations (8.88%) among the altered genes; the most frequently involved gene was RNF168, followed by FBXW7 and HERC2 (10.78%, 38.6%, and 17.58%, respectively; Table 1). BRCA1 somatic mutations are newly recognized as clinically significant factors of genomic instability (39). The highest rates of BRCA1 mutations tend to cause somatic loss-of-function mutations in ovarian cancer due to massive genomic instability and tumor formation (40, 41). BRCA1-mutated tumors regulate EMT in breast cancer cells and cancer metastasis, the cause of death from all cancers (42).
RNF126 positively regulates HR through BRCA1 transcription by interacting with E2F1 (20). RNF126/E2F1 facilitates the transcription of CHK1, which is required in repairing replication stress-induced DSB (43). RNF126 promotes NHEJ by K48-linked ubiquitination of Ku70/Ku80 and loosening of the Ku70/80 heterodimer from DNA DSB sites. Interestingly, the release of the Ku70/80 heterodimer from DNA DSB sites facilitates the accomplishment of DSB repair (21). Finally, RNF126 recruitment at DSB sites occurs in an ATM- and RNF8-dependent manner and RNF126 directly ubiquitinates RNF168, which leads to the inhibition of RNF168-mediated ubiquitination of γH2AX (22). By contrast, RNF126 negatively regulates the foci formation of 53BP1, γH2AXub, RNF168, BRCA1, and RAP80 IR-induced DSB (43). The cBioPortal database showed significantly increased somatic mutation of RNF126 in sarcoma. Numerous examples of high RNF126 expression in sarcoma and brain lower grade glioma have been documented. Recently, studies have revealed an oncogenic role of RNF126 in various cancers, such as breast, gastric, prostate, and ovarian cancers (43-46). RNF126 was overexpressed in human colorectal cancer specimens, which was closely related to tumor size, lymph node metastasis, and the poor survival of colorectal cancer (45). Furthermore, RNF126, as an oncogene, causes ubiquitination and the proteasome pathway degradation of PTEN in bladder cancer and, consequently, induces pulmonary metastasis in bladder cancer (43).
RNF138 regulates HR, which is initiated by DNA end resection, and this mechanism is associated with the ubiquitylation of Ku80 (19). RNF138 ubiquitylates Ku80 following HR repair pathway utilization. Once Ku is removed, RNF138 recruits RPA loading and CtIP and EXO1; consequently, the DNA ends produce ssDNA overhangs (47). Highly expressed RNF138 increased cell viability and postponed cell cycle progression and cisplatin resistance by inhibiting Chk1 signaling. RNF138 modulates cisplatin resistance in gastric cancer cells. Furthermore, cisplatin resistance leads to a promising drug target to explore chemotherapy failure (47). The cBioPortal database showed significantly increased somatic mutation of RNF138 in uterine corpus endometrial carcinoma and its high amplification in ovarian serous cystadenocarcinoma. According to a recent study, RNF138-mediated rpS3 ubiquitination induces nuclear translocation of rpS3, thereby led to radioresistance in glioblastoma cells (48).
Activation of ATM induced by DNA DSBs recruits the ubiquitin ligases, RNF8, and RNF168, which induce chromatin-ubiquitinating histone γH2AX near DSB sites. The two E3 ubiquitin ligases RNF8/RNF168 ubiquitylate γH2AX K13/K15 upon genotoxic stress (11). This complicated ubiquitin network regulates the foci formation to promote the recruitment of DSB factors, such as BRCA1 and 53BP1, required for downstream signaling (49, 50). Recently, the RNF168/UbcH5c complex was shown to promote K27 ubiquitination of nucleosomal H2A/γH2AX. H2A/γH2AX K27 ubiquitination is required to activate DSB factors, such as 53BP1, RAP80, RNF168, and RNF169 (51). RNF168 is aberrantly overexpressed in many cancers, and one reason for this is the presence of mutations in DSBs. RNF168 was overexpressed in various cancers, such as lung, esophageal, and ovarian cancers. RNF168 was the amplified gene in nearly all cancers, with 31.62% of lung cancer showing a few types of RNF168 mutation and were consistent with mutations that influenced DNA repair. Overexpression of RNF168 and activated 53BP1 recruitment induce mutagenic NHEJ at the HR repair, resulting in reinforced genomic instability and radio-resistance in breast cancer cell lines (52). Of 27 cancer types with DSB-related RNF168 has been confirmed to be a mutation in 20 cases (74.07%) in various cancer cells (Table 1). Moreover, we examined the commonness of NHEJ pathway alterations by cancer type and identified the most frequently mutated gene within the signaling pathway for each cancer type. For example, RNF168 exhaustion inhibits the growth of BRCA1/BRCA2-deficient breast and ovarian cancer cells, resulting in DSBs, senescence, and subsequent cell death (53).
RNF169 has a similar domain architecture to RNF168. Despite this, the affinity of RNF169 for γH2A K13/K15 is higher than that of RNF168 (54). The UDM2 domain of RNF169 prevents IR-induced 53BP1 and regulates RNF168-dependent signaling by interrupting DNA DSB-induced ubiquitylation of γH2AK13/K15ub (55). Remarkably, RNF169 is necessary for the recruitment of various DDR proteins, including BRCA1, RAP80, and RAD18, near DSB sites (56). Gain-of-function mutations were amplified in RNF169 in esophageal cancer. The cBioPortal database showed that RNF169 amplification was significantly associated with various cancers, such as esophageal, ovarian, and breast cancer. USP7 deubiquitylates and stabilizes RNF169 through nuclear import in breast cancer (57).
SKP2, also known as F-Box/LRR-repeat protein 1 (FBXL1) or p45, is one of the members of the F-box protein family and is involved in ubiquitination, cell cycle regulation, and signal transduction regulated by the SKP2-SCF complex (58, 59). SKP2 affects MRN (Mre11-Rad50-Nbs1) complex-mediated ATM activation by triggering K63-linked NBS1 at DSB sites (60). The cBioPortal database showed significantly increased SKP2 amplification in cancers, such as lung squamous cell carcinoma, lung adenocarcinoma, and bladder urothelial carcinoma (60). Numerous examples of high SKP2 somatic mutation in uterine corpus endometrial carcinoma have been documented. Overexpression of SKP2 plays a multifaceted role in cancer by enhancing the interaction between ATM and the MRN complex. SKP2 remarkably promotes PDCD4 phosphorylation, ubiquitination, and degradation. In other cancer cells, SKP2 induces cell proliferation, cell apoptosis, and inhibition of enhanced PDCD4 during DSB repair in breast cancer (61).
FBXW7 is established as the substrate recognition subunit for the SCF E3 ubiquitin ligase (62, 63). Activated ATM phosphorylates S26 of FBXW7 to promote its recruitment to DSB sites. In the DSB pathway, Ku70/80 quickly binds to DNA ends and serves as the scaffold to recruit the core NHEJ machinery. Subsequently, The DNA-PKcs, XRCC4, Artemis, XLF, and LigIV are independently recruited to the Ku-DNA complex (7-9). FBXW7-mediated polyubiquitination of K63-linked XRCC4 on K296 promotes NHEJ, expedites NHEJ complex formation, and promotes NHEJ repair (64). Highly mutations have been found in FBXW7 (>10% of samples) in at least three tumor types: uterine carcinosarcoma, uterine corpus endometrial carcinoma, and colorectal adenocarcinoma. Despite numerous studies, the prognostic importance of FBXW7 mutations in uterine carcinosarcoma remains uncertain. FBXW7 regulates hematopoietic stem cell (HSC) inactivity and self-renewal (65, 66). FBXW7 regulates the degradation of cyclin E, upregulates Myc overexpression in HSC, and causes defective HSC self-renewal after hematologic stress (67, 68). Decreased cyclin E causes extraordinary cell cycle control, epithelial cell proliferation, impaired genome instability, and erythroid cell differentiation (69, 70).
The HERC2 E3 ubiquitin ligase interacts with NEURL4 and MDM2 to regulate p53-dependent gene stability in ATM- and ATR-induced DNA DSB repair (71, 72). The DSB repair pathway begins with the recognition of the sites of DNA damage by the MRN complex, then NBS1 recruitment, which promotes the binding of ATM to the DSB site, in turn, leading to phosphorylation of γH2AX S139 and bind MDC1 (73). The γH2AX/MDC1 complex promotes the recruitment of HERC2 and RNF8. HERC2 promotes RNF8 oligomerization and expedites the formation of the HERC2-MDC1-RNF8 complex, following RNF8-mediated recruitment of RNF168 (13). In addition to regulating the MDM2-p53 transcriptional pathway, HERC2 can inhibit potentially oncogenic mutations from being passed to daughter cells by regulating DNA DSBs during the S and G2-M phases of mitosis (74, 75). The cBioPortal database showed significantly increased overexpression and mutation of HERC2 in cancers, such as skin cutaneous melanoma, uterine corpus endometrial carcinoma, and lung adenocarcinoma. Furthermore, amplification of HERC2 has been discovered in several human cancers, such as cutaneous cancer, uveal melanoma, and non-small cell lung cancer (76). Additionally, the types of cancer associated with HERC2 include pheochromocytoma, paraganglioma, and T-cell prolymphocytic leukemia (77, 78).
Human WWP2 contains a HECT domain E3 ligase for NHEJ repairs. RNA polymerase II (RNAPII) is recruited to DNA double-strand breaks for inhibiting of transcription. As WWP2 promotes the degradation of RPB1 of RNAPII subunit through K48-linked ubiquitination regulates DNA-PK-dependent transcription arrest and repair at DNA breaks. The cBioPortal database showed significantly increased somatic mutation of WWP2 in cancers, such as uterine corpus endometrial carcinoma. Numerous examples of high expression of WWP2 in breast cancer and liver cancer have been documented. WWP2 has key roles in the evasion of apoptosis and the over-proliferation of liver cancer; WWP2 overexpression was upregulated in cancer tissues. WWP2 knockdown remarkably decreased the proliferation, caused apoptosis, and induced cell-cycle arrest in Huh7 and BEL-7404 cells (79-82). Moreover, WWP2 is highly expressed in hepatocellular carcinoma and activation of AKT signaling to increase tumor metastasis (83). DNA damage enhances the differentiation of glioma stem cells by WWP2. SOX2 regulates to maintain the self-renewal potential of glioma stem cells in glioblastoma. WWP2 ubiquitylates SOX2 through its HECT domain to enhance Sox2 ubiquitination, thus stabilizing glioma stem cell maintenance (84).
Over the last decades, DSB-associated E3 ligases have been continuously studied for their importance of somatic mutations in cancer cells, but they have not yet solved all the mechanisms. In this study, we revealed that genomic instability caused by DSB-related E3 ligases is manifested as distinct cancer somatic mutations in RNF20/40, BRCA1, RNF168, FBXW7, HERC2, and WWP2 and the expression levels of these genes. Based on the mutation expression of DDR-related E3 ligase through the TCGA, associated with gene instability and cancer. Most of the DSB-related E3 ligases have known functions, but the physical functions in cancer somatic mutations and over-amplification have not been elucidated. It is necessary to study genomic instability through mutations found in patients with cancer. Although many studies have been conducted on the association between gene amplification and cancer, research on the association with mutations is still lacking. The realization that DNA DSB repair pathways, which protect the genome against DSB-related gene changes, can serve as essential targets for cancers is highly encouraging. DSB-related genes are highly expressed in particular cancers, induce radioresistance or chemoresistance. Consequently, strategies utilizing the DNA DSB repair pathways as targets for anticancer treatment can result in the development of potent candidates for therapeutics. Research on gene amplification and mutation-induced gene instability in cancer cells is continuously needed based on the TCGA database and samples from patients with cancer.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (No. NRF-2022R1H1A2091883, NRF-2019R1A2C1007197).
The authors have no conflicting interests.
Analysis of data in The Cancer Genome Atlas (TCGA) cBioPortal indicates genetic alterations of E3 ligase in several types of cancer
|E3 ligase||Related E3 ligase in cancer||Mutation (%)||Mutation sites||Amplification (%)||Reference|
||UCEC: genes altered in 4.91% of 529 cases 3.97% (21 cases)||
||OSC: genes altered in 4.97% of 584 cases 4.97% (29 cases)||(85, 86)|
||UCEC: genes altered in 7.18% of 529 cases 6.81% (36 cases)||
||Adrenocortical carcinoma: genes altered in 3.3% of 91 cases 3.3% (3 cases)||(31)|
||UCEC: genes altered in 9.26% of 529 cases 8.88% (47 cases)||
||Pancreatic adenocarcinoma: genes altered in 3.26% of 184 cases 2.17% (4 cases)||(49, 87)|
||Skin cutaneous melanoma: genes altered in 2.7% of 444 cases 2.25% (10 cases)||
||Sarcoma: genes altered in 8.24% of 255 cases 5.49% (14 cases)||(20, 22, 88)|
||UCEC: genes altered in 3.02% of 529 cases 2.65% (14 cases)||
||OSC: genes altered in 5.14% of 584 cases 4.11% (24 cases)||(19, 89)|
||UCEC: genes altered in 10.78% of 529 cases 4.35% (23 cases)||
||Lung squamous cell carcinoma: genes altered in 31.62% of 487 cases 30.18% (147 cases)||(54, 90)|
||UCEC: genes altered in 4.35% of 529 cases 3.02% (16 cases)||
||Esophageal adenocarcinoma: genes altered in 8.79% of 182 cases 6.59% (12 cases)||(91, 92)|
||UCEC: genes altered in 6.24% of 529 cases mutation 3.21% (17 cases)||
||Lung squamous cell carcinoma: genes altered in 10.47% of 487 cases 10.06% (49 cases)||(60)|
||Uterine carcinosarcoma: genes altered in 38.6% of 57 cases 38.6% (22 cases)||
||Sarcoma: genes altered in 3.53% of 255 cases 2.75% (7 cases)||(64)|
||UCEC: genes altered in 18.53% of 529 cases 17.58% (93 cases)||
||Sarcoma: genes altered in 5.1% of 255 cases 1.96% (5 cases)||(13)|
||UCEC: genes altered in 7.37% of 529 cases 5.29% (28 cases)||
||Cholangiocarcinoma: genes altered in 5.56% of 36 cases 2.78% (1 case)||(10)|