The E3 Ligases in Cervical Cancer and Endometrial Cancer

Simple Summary We summarized and cataloged the E3 ligases involved in endometrial carcinoma (EC) and cervical cancer (CC). Finally, an overview of the current drugs that target the ubiquitination process to rescue patients with EC and CC is presented to provide researchers with important research ideas that can be applied to clinical treatment. Abstract Endometrial (EC) and cervical (CC) cancers are the most prevalent malignancies of the female reproductive system. There is a global trend towards increasing incidence and mortality, with a decreasing age trend. E3 ligases label substrates with ubiquitin to regulate their activity and stability and are involved in various cellular functions. Studies have confirmed abnormal expression or mutations of E3 ligases in EC and CC, indicating their vital roles in the occurrence and progression of EC and CC. This paper provides an overview of the E3 ligases implicated in EC and CC and discusses their underlying mechanism. In addition, this review provides research advances in the target of ubiquitination processes in EC and CC.

Increasing evidence indicates that critical protein ubiquitination in various signaling pathways plays a crucial role in the development of EC and CC. This manuscript summarizes the relationship between ubiquitination and some important EC and CC-related signaling pathways, such as the p53, the Nuclear Factor Kappa-B (NF-κB), the epidermal growth factor receptor (EGFR), the Wnt/β-catenin, and TGF-β/Smad pathways ( Figure 2, Table 1).  3.1. p53 Pathway p53 dysfunction in malignant tumors is mainly due to the inactivation of the p53 protein by mutations in binding proteins or TP53. p53, a tumor suppressor protein, is a potent transcription factor that promotes cell cycle arrest, senescence, and apoptosis by regulating more than 100 target genes in response to various stimuli [75]. Although p53 regulates the expression of several tumor suppressor proteins, it is usually retained in an "off" state because of its rapid turnover [76]. Monoubiquitination of the C-terminal domain of p53 acts not only as a scaffold for polyubiquitination but also as a signal for the nuclear export of p53, which is the foremost step in limiting p53 function [77][78][79]. In the cytoplasm, multiple E3 ligases lead to the ubiquitination of p53 and the degradation of the 26S proteasome [80]. Thus, the critical role of E3 ligases in the p53 signaling pathway associated with EC and CC is comprehensively summarized.

MDM2
Murine double minute 2 (MDM2) is a well-known oncogenic protein, which is composed of an N-terminal p53-binding domain, nuclear localization signal (NLS), nuclear export signal (NES), acidic domain, zinc finger domain, and C-terminal RING finger domain [81]. The RING finger domain, which is located in the C-terminal, is the main functional domain [82]. MDM2 acts as a cancer-promotor in EC and CC by regulating the stability of p53 [57,58]. MDM2 not only plays a critical role in recognizing the N-terminal trans-activating domain (TAD) of p53 but also acts as a p53 transcriptional activation repressor [83]. Furthermore, MDM2 phosphorylation appears to be a key step in mediating p53 degradation and preventing its ubiquitination [84,85]. The mechanism associated with MDM2 and p53 degradation is best shown in mouse models, in which the inactivation of p53 completely rescues the embryonic mortality of MDM2 when its function is lost [86]. The prototype small molecule CP-31398, a synthetic styrene quinazoline, can inhibit EC proliferation and migration by down-regulating MDM2 expression and stabilizing p53 activity [87]. Previous studies have shown that CP-31398 blocks the migration and invasion of hepatocellular carcinoma (HCC), colorectal cancer, and PCa caused by p53 deficiency both in vitro and in vivo [88][89][90]. Although there are few studies on the relevant mechanisms of MDM2 in promoting cancer in EC and CC, the existence of substrates other than p53 or molecules that indirectly interact with p53 requires further exploration. The role of E3 ligases in the p53 pathway, the Nuclear Factor Kappa-B (NF-κB) pathway, the EGFR pathway, the Wnt/β-catenin pathway, the TGF-β/Smad pathway in endometrial carcinoma (EC) and cervical cancer (CC). The p53 signaling pathway: E3 ligases murine double minute 2(MDM2), E6AP, and TRIM65 directly bind to p53 and promote ubiquitination degradation of p53. p53 protein, in turn, induces MDM2 to regulate itself through self-ubiquitination. Furthermore, RBBP6 promotes the phosphorylation of c-Jun NH2-terminal kinase (JNK) to regulate the expression of p53. In CC cells, FHL2 binds to IER3 and MDM2 to form a ternary complex that allows for efficient MDM2-mediated degradation of IER3 by enhancing the association between MDM2 and IER3. NF-κB pathway: E3 ligase TRIM22 directly binds to nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Reducing the phosphorylation of p65 and IκBα reduces NF-κB signal transduction. Epidermal growth factor receptor (EGFR) pathway: makorin RING finger protein 1 (MKRN1) can be phosphorylated by the EGFR/PI3K/AKT pathway to continuously degrade PTEN protein in a K48-dependent manner. Wnt/β-catenin pathway: RNF43 directly interacts with Wnt and promotes its degradation, thereby inhibiting continuous activation of the Wnt/β-catenin pathway. TGF-β/Smad pathway: Activated TGF-β ligand interacts with two transmembrane receptors TβRI and TβRII, to form a tetrameric complex. Constitutively activated TβRII phosphorylates and activates TβRI, which directly phosphorylates downstream transcription factors Smad 2 and Smad 3 through its serine/threonine kinase activity. Activated Smad2 or Smad3 can bind to Smad4, migrate to the nucleus, and bind to different TFs to achieve excessive activator or suppressor activity. SCF Skp2/Cks1 directly interacts with p27 and ubiquitinate it.

E6AP
Almost all cervical precancerous and malignant squamous cell lesions are associated with HPV infection. High-risk HPV-expressed E6 oncoprotein can recruit E6AP and subsequently induce 26S proteasome degradation of p53, which is a key factor in CC cell transformation [59]. E6AP belongs to the HECT family, with HECT domains involved in binding substrates and distinct N-termini that mediate substrate specificity [91]. The E6/E6-AP complex regulates various HPV-induced oncogenic proteins through proteasome [92]. These oncogenic proteins include E6 targeting protein 1(E6TP1), Bcl-2 antagonist killer (Bak), and cellular myelocytomatosis oncogene(c-Myc), which will continue to be extended as more research is conducted on the role of E6AP [14]. p53 mutations frequently occur in cancer cells and are concentrated in the DNA-binding domain [93]. A study by Li et al. emphasized that the tumor suppressor function of wild-type p53 could be established using a p53 mutant [59]. A recent study showed that E6 phosphorylation inhibits the transcriptional activity of p53 [94]. The PSD-95/Dlg/ZO-1 binding motif (PBM) of the C-terminal of E6 contains a well-characterized phosphate receptor site that, in the absence of E6AP, leads to a significant increase in p53 levels and phosphorylated E6 (pE6) levels, which in turn inhibits p53 transcriptional activity [59]. Thus, a negative feedback loop is formed.
TRIM65, which belongs to the RING E3 ligase family, is a 517 aa length protein [49]. TRIM65 is widely associated with white matter lesions, innate immunity, tumors, and other diseases [49]. The study by Wang et al. reported that TRIM65 is dramatically overexpressed in human CC tissues [13]. In vitro experiments have demonstrated that TRIM65 can interact with p53 and promote p53 ubiquitination, thereby inhibiting the apoptosis of CC cells. In addition, the overexpression of TRIM65 can promote mTOR phosphorylation, and this process will block the degradation of the pathogen and decrease autophagy-related apoptosis.

RBBP6
Retinoblastoma-binding protein 6 (RBBP6) belongs to the RING E3 ligase family and is closely related to apoptosis, cell cycle regulation, and tumorigenesis [95,96]. Three subtypes can be encoded by the RBBP6 gene: Rb, the RING domain, zinc, and p53 binding domains comprise subtype1 and 2, which serve as E3 ligases; subtype 3 contains a domain with no name (DWNN) domain, which possesses the ability of ubiquitin-like modification [97,98]. RBBP6 regulates p53 protein levels and activity through three pathways. First, RBBP6 interacts directly with p53 to regulate gene transcription [95]. Second, RBBP6 promotes apoptosis through ubiquitination and degradation of Y-box binding protein 1 (YB-1), which can serve as a transcription factor and associate with the suppression of p53 [99]. Third, RBBP6 interacts with the E3 ligase MDM2 and enhances its ability to mediate p53 ubiquitination and proteasome degradation [100]. RBBP6 was overexpression in CC tissues.
In vitro experiments showed that when the expression of RBBP6 was upregulated, the c-Jun NH2-terminal kinase (JNK) phosphorylation increased, and the transcription of p53 was inhibited, leading to a significant increase in the viability, migration, and proliferation in CC cells [60].

NF-κB Pathway
NF-κB belongs to the transcription factor family, which is expressed in almost all animal cell types and activated by various signaling proteins. It is a major pro-inflammatory signaling pathway required for the rapid activation of immune cells [101,102]. The NF-κB pathway is associated with carcinogenesis, inhibition of apoptosis, and promotion of resistance to anticancer drugs [103]. Various stimuli can activate the classic NF-κB pathway, such as TNFα, LPS, IL-1β, IL-1R, TLR, TNFR, and antigen receptors, which in turn promote the activation of IκB kinase β (IKKβ). IKKβ activation can promote IκBα phosphorylation, which mediates IκBα ubiquitination. The release and translocation of NF-κB dimers promote nuclear drive target genes transcription [104]. Current studies have found that NF-κB induced inflammation promotes the development of EC. In addition, NF-κB signaling pathways are regulated by various E3 ligases, such as CUL3 SPOP , TRIM22, and TRIM25.  [105]. Substrates that can be ubiquitinated in human cells include death dome-associated protein (DAXX), phosphatase (PUC), steroid receptor coactivator(SRC-3) and transcriptional regulator (CI/GLI), estrogen receptor-α (ERα), and BRD2, BRD3, and BRD4 proteins (BETs) [25,106]. SPOP is mutated in 8.7% of patients with EC, based on the data available in the cBioPortal portal (https://www.cbioportal.org (accessed on 23 September 2022)). These mutations occur mainly outside the cleavage on the substrate-binding surface in the MATH region [107]. ERα, encoded by the ESR1 gene, is highly overexpressed in hormone-responsive cancers, such as ovarian, endometrial, and breast cancers [108]. A previous study showed that CUL3 SPOP interacts with ERα and triggers its ubiquitination and degradation, thus suppressing the occurrence and progression of EC. Then, this process inhibits the cancerpromoting effects of ERα in EC [105]. However, EC-related CUL3 SPOP mutants are flawed in the ubiquitination of ERα [105]. Another study showed that CUL3 SPOP could ubiquitinate and degrade BETs proteins, particularly BRD3, in PCa and EC [106]. Controversially, PCaassociated CUL3 SPOP mutations inhibit BET protein degradation, whereas EC-associated CUL3 SPOP mutations potentiate BET protein degradation [106,109]. Currently, the functional gain mechanism and precise structural basis of EC-related CUL3 SPOP enhancing BET protein degradation need to be further elucidated. Death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) can act as a potential positive regulator of apoptosis. DRAK1 depletion enhances TRAF6 protein expression, which ultimately activates NF-κB signaling and promotes oncogenesis in CC cells [61]. Pang et al. demonstrated that the down-regulation of DRAK1 expression is usually accompanied by paclitaxel resistance in CC cells. Furthermore, increasing the protein expression level of DRAK1 could make CC cells more sensitive to carboplatin and paclitaxel [110]. Mechanistically, CUL3 SPOP mediates DRAK1 proteasome degradation through K48-linked polyubiquitin, leading to increased TRAF6 levels. TRAF6 then promotes CC progression by activating the NF-κB signal pathway.

TRIM22
Tripartite motif-containing 22 (TRIM22) contains three main domains: coiled-coil, B-box, and a RING finger [62]. TRIM22 also associates with various biological processes in transcription regulation [111] TRIM22 expression is downregulated in EC tissues, which is associated with high malignancy and poor prognosis [112][113][114]. Zhang et al. confirmed that TRIM22 could induce and promote nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) expression, reduce the phosphorylation of P65 and IκBα, resulting in reducing NF-κB signal transduction, and ultimately inhibit EC development and progression [62]. NOD2 belongs to the NOD-like receptors (NLRs) family that is stimulated by TLR to induce IRF4, which subsequently inhibits NF-κB activation through interactions with MyD88 or TRAF6, enabling NOD2 to inhibit NF-κB signaling [115,116]. TRIM22 can be used as a progesterone target gene to increase EC patients' prognosis [117]. In conclusion, TRIM22 is a significant predictor for prognostic and also is a potential therapeutic target for patients with EC. Comprehensive and in-depth studies on the potential mechanism of TRIM22 downregulation in patients with EC are urgently required.

TRIM25
TRIM25 is structurally similar to TRIM22 and belongs to the TRIM protein superfamily [118]. TRIM25 has been confirmed to contribute to the aggressiveness of various cancers. For example, in breast cancer cells, TRIM25 degrades the cell cycle checkpoint protein 14-3-3σ via UPS. Thereby, this process will promote the formation of xenograft tumors in mice [119]. TRIM25, an interferon (IFN) responsive gene, modulates the NF-κB pathway [120,121]. Sato et al. demonstrated that TRIM25 degrades 14-3-3σ protein through ubiquitination, thereby enhancing NF-κB signaling and the related factors which can promote the development of EC [63]. The expression level of TRIM25 is reduced in EC tumors and positively related to the ERα expression, suggesting that the function of TRIM25 on EC may be related to ERα signal transduction [122,123]. However, in vitro TRIM25 downregulation reduced EC cell migration in ERα+ and ERα-cell lines, regardless of estrogen treatment. Therefore, the natural role of TRIM25 in EC requires further investigation.

EGFR Pathway
Epidermal growth factor receptor (EGFR) is a member of the ErbB family and an important signaling pathway activating receptor on the cell membrane surface. EGFR dimerization activates PI3K/AKT and other signaling pathways through the phosphorylation cascade to play biological functions. Phosphatase and tensin homolog (PTEN), as a tumor suppressor, is a potent inhibitor of the PI3K/AKT signaling pathway because PTEN promotes the dephosphorylation of phosphatidylinositol-3, 4, 5-triphosphate (PIP3) to phosphatidylinositol-4, 5-diphosphate (PIP2) [124]. The crosstalk between the phosphorylation cascade and ubiquitination affects various cellular physiological processes [125]. For example, Li et al. found that the EGFR/PI3K/AKT/makorin RING finger protein1 (MKRN1)/PTEN axis plays an important role in the occurrence and development of CC [64]. MKRN1 is an E3 ubiquitin ligase that can be more stable via being phosphorylated by pAKT at S109 [64], while phosphorylated MKRN1 persistently induces polyubiquitination and degradation of the K48-linked PTEN [64].

Wnt/β-Catenin Pathway
The Wnt signaling pathway plays a role in a wide range of animal cells and is a highly conserved signaling pathway in species genetics. Persistent abnormal Wnt activation or abnormal accumulation of β-catenin is a key process in tumorigenesis [126,127]. It has been demonstrated that E3 ligase promotes EC development by labeling critical molecules of this pathway, such as RNF43 [128,129].
RNF43 is a type I integral membrane protein containing a RING finger domain with the vast majority of its aa motifs located in the intracellular region [130]. RNF43 is a negative regulator of the Wnt signal. It and the related side family homolog ZNRF3 can ubiquitinate Wnt receptors to function [128,129]. By querying the COSMIC cancer database, we find that the most common RNF43 mutations are nonsense or shift mutations. In addition, the most common truncating mutation is G659Vfs*41. The frequency in EC is about 5-8% [65]. Treatment for patients with RNF43 mutated EC mainly focuses on sustained inhibition of Wnt signaling by inhibiting the production of Wnt ligands. However, no study has demonstrated the feasibility of such a scheme.

TGF-β/Smad Pathway
The transforming growth factor-β (TGF-β) signaling pathway plays an important physiological function in maintaining normal cellular physiology and promoting embryonic maturation and development, among other processes. Abnormal signaling of TGF-β, a potent endogenous proliferation inhibitor, occurs in the development of many human type I EC [131]. Activated TGF-β ligand interacts with TβRI and TβRII, which can form a tetrameric complex. Constitutively activated TβRII phosphorylates and activates TβRI, which directly phosphorylates downstream transcription factors Smad2 and Smad3 through its kinase activity [132]. Active Smad2 or Smad3 can bind to Smad4, then migrate to the nucleus. They also can bind to different transcription factors (TFs) to achieve excessive activator or suppressor activity [133]. In general, the antitumor effects can be achieved by inactivating essential components of this signaling pathway, such as p27kip1 (p27) [134].
APC and the SCF Skp2/Cks1 complex are two major E3 ligases that can regulate cell cycle progression [135,136]. In normal endometrial epithelial cells, TGF-β promotes APC binding to Cdh1, forming an E3 ligase complex capable of ubiquitination of SCF Skp2/Cks1 , thereby reducing the degradation of nuclear p27, which then inhibits cyclin-dependent kinase 2(Cdk2) to prevent EC cell growth [137]. Sustained ubiquitination of p27 by SCF Skp2/Cks1 is an early causative event of type I EC. Furthermore, estrogen promotes phosphoryla-tion of the Thr187 site of p27 in a MAPK/erk2-dependent manner, which is required for SCF Skp2/Cks1 to recognize and ubiquitinate p27 [66]. In contrast, progesterone inhibits estrogen-induced endometrial proliferation during the menstrual secretory phase [138]. Therefore, progesterone has been identified as a treatment drug for type I EC.

SCF FBXO2 and SCF FBXW7
The F-box protein is an adaptor of the Skp1-CUL1-F-box ubiquitin ligase complex, which is characterized by approximately 40 aa motifs [139]. It is involved in cell growth, development and differentiation, signaling responses, and cell survival and death by degrading cancer-related regulatory proteins [140]. The F-box protein dysregulation can lead to sleep loss, diabetes, Parkinson's disease, and infectious diseases [141][142][143]. Currently, it can be divided into three families according to the differences in recognizable domains: the F-box and WD40 domain (FBXW) family contains the WD-40 domain, the F-box and Leu-rich repeat (FBXL) family is rich in leucine repeats, and the remaining family belongs to the F-box only (FBXO) family. Notably, F-box proteins are associated with various biological pathways, with the most well-defined biological roles and properties of EC being FBXO2 and FBXW7. SCF FBXO2 is predominantly present in neurons and mediates related glycoproteins ubiquitination, thereby regulating their associated functions and safeguarding the normal function of neuronal cells [144,145]. Furthermore, the Fox-associated domain (FBA) is crucial for its glycoprotein-recognizing activity. It can bind to the N-linked highmannosylglycan fraction marked by glycoproteins [145]. Recently, the roles of SCF FBXO2 have been experimentally confirmed in diverse cancers. For instance, SCF FBXO2 regulates the EMT in gastric cancer. Of concern, in colorectal cancer, SCF FBXO2 has been used to predict poor prognosis [146,147]. Che et al. demonstrated that overexpression of FBXO2 patients with EC is associated with various poor clinical indicators. SCF FBXO2 can interact with fibrillin, which is considered an essential tumor suppressor through the FBA domain and mediates its ubiquitination and degradation [67]. In addition, their data suggest that the abnormal expression of SCF FBXO2 is associated with the cell cycle and autophagy signaling pathways. SCF FBXW7 , also known as SCF FBXW7 and Cdc4, acts as a tumor suppressor and is associated with at least 35 tumor types [14]. FBXW7 can be divided into three main isoforms according to their N-termini difference. FBXW7α localizes to the nucleoplasm, which is the most abundant isoform. FBXW7β localizes to the cytoplasm. FBXW7γ isoforms localize to the nucleolus [148,149]. SCF FBXW7 maintains normal cellular biological functions in normal cells by degrading several carcinogenic regulators that target proliferation, growth, and apoptosis, including Notch 1, cyclin E (CCNE1), c-Myc, Aurora kinase A, and Jun [150]. Mutations in FBXW7 is approximately 6% of all kinds of cancers, the most common of which is T-cell acute lymphoblastic leukemia (T-ALL; 31%) [151,152]. In addition, a novel nonsense mutation has been confirmed to be associated with colorectal cancer. In EC, missense mutations are the predominant form of FBXW7 mutations (about 16%) [153].
Even though the frequency of FBXW7 mutations in EC is high, the individual context and molecular consequences of such mutations remain poorly understood. Kuhn et al. proved that the existence of FBXW7 mutation and CCNE1 amplification raised the level of CCNE1 protein, thereby promoting cell proliferation [68]. Therefore, future work should explore more molecular biological effects of EC-associated FBXW7 mutations and determine whether FBXW7 mutations can be used as precision therapeutic targets to develop targeted therapeutic drugs. This work may be critical for improving patient care with clinically aggressive EC.

APC Cdc20
Evidence has emerged that APC is the predominant driver in the control of cell cycle progression [154]. The APC core is interlinked with the activator cell division cycle 20 ho-molog (Cdc20), allowing it to form a complex capable of biological function [155]. Emerging evidence suggests that Cdc20 recruits its substrates through different motifs, such as the destruction box (D-box), TEK, and the newly identified ABBA [156][157][158][159]. APC Cdc20 is known to exhibit carcinogenic effects in various cancers, including breast, hepatocellular, lung, bladder, and endometrial [160]. The gene encoding the 17-β-estradiol (E2) oncoprotein is often inactivated in CC cells infected with high-risk HPV-18. In addition, E2 can inhibit two viral oncogenes' expression, E6 and E7, which are the main steps in the HPV-induced transformation of CC cells [161]. Although E2 can interact with Cdc20 and Cdh1, two co-activators of APC, E2 is not ubiquitinated by APC. Instead, E2 can inhibit APC function, allowing substrates accumulation, such as SCF Skp2/Cks1 . In the late G1 phase, SCF Skp2/Cks1 directly interacts with E2 and mediates proteasomal degradation [162]. Simultaneously, it also promotes E6 and E7 expression and induces HPV-infected cells' early entry into the S phase. Yu et al. showed that the HPV16 E7 oncoprotein can highly express and inhibit EMI1 degradation in mitotic cells by promoting the APC inhibitor EMI1. Overexpression of EMI1 suppresses substrate degradation and prophase delay of mitosis [163]. Long noncoding RNA (lncRNAs) plays an essential role in cells by influencing protein ubiquitination [65,[164][165][166][167]. Studies report that lncRNA-ZXF1 has been shown to be an important regulator of EC development [69]. In normal endometrial cells, lncRNA-ZXF1 can interact with P21 to stabilize the protein level of P21 and avoid its degradation by APC Cdc20 [69]. Therefore, lncRNA-ZXF1 may be an important prognostic indicator for precision therapy because of its tumor suppressor effect in EC.

HUWE1
HUWE1, an E3 ligase with a catalytic HECT domain and a substrate-binding loop. The number of substrates that can be ubiquitinated by HUWEI1 has exceeded 40. It is closely related to cell stress responses, cell statue, signal transduction, inflammasome activation, and other cellular processes [168]. Comprehensive molecular studies have shown that HUWE1 can target various substrates, including c-Myc [169]. In CC, the expression level and activity of HUWE1 are significantly down-regulated, which promotes c-Myc expression and cell propagation [70]. Specific inhibitors of c-Myc against various human tumors are in stage I/II clinical trials. However, because the discovery of c-Myc inhibitors is difficult, BET inhibitors are currently used as an alternative strategy in preclinical studies. For example, the BET inhibitor GS-626510 could effectively inhibit xenograft tumor growth in vivo [70].

Cullin2 E7
High-risk HPV E7 is closely related to the development of CC. Typically, E7 exerts its oncogenic potential by inactivating the normal function of tumor oncogenic factors [170]. Other properties of E7 suggest its involvement in virus-induced cell transformation. Rb protein plays an important role in the development of CC, and it can be degraded by E7-mediated ubiquitination. Similar to E6-mediated p53 ubiquitination degradation, E7 recruits E3 ligase to target Rb leading to its ubiquitination and degradation in CC [171]. At present, Cullin2 is the only E3 ligase that can bind to E7 to form a complex, but there is no evidence in vitro [172]. Notably, Wang et al. found that a novel Rb E3 ligase (NRBE3) promotes Rb degradation via K48-dependent ubiquitination in vivo and in vitro [173]. Whether NRBE3 can bind to HPV E7 to induce Rb degradation in CC is worthy of further exploration.

MDM2
Immediate early response 3 (IER3) can rapidly induce apoptosis of CC cells in various ways [174][175][176][177]. In CC cells, MDM2 directly binds with IER3 and mediates its K60 polyubiquitination [71]. Four and a half LIM domain-containing protein 2 (FHL2) is a multifunctional protein associated with various cellular biological processes, including gene manifestation regulation, cell statue, propagation, differentiation, adhesion, and migration [178][179][180]. Due to its structure, FHL2 acts as a binding site that interacts with different proteins and forms complexes [179,181]. In CC cells, FHL2 binds to IER3 and MDM2 to form a ternary complex that allows the efficient MDM2-induced degradation of IER3 by enhancing the association between MDM2 and IER3 [71].

RNF114 and Smurf1
RING finger 114 (RNF114) and Smurf1 are members of the E3 ligase family. Studies have demonstrated that RNF114 can positively regulate the activity of poly (ADP-ribose) polymerase 10 (PARP10) through K27-linked polyubiquitination in CC [72]. Zhao et al. identified PARP10 as a tumor metastasis suppressor and demonstrated that PARP10 interacts with Aurora A and then inhibits its kinase activity, thereby regulating Aurora A downstream signaling to inhibit the EMT process in tumor cells and tumor metastasis [72,182]. Interestingly, the G888W mutant of PARP10 blocks its interaction with RNF114 and abolishes the enzymatic activity of PARP10 [183]. Many studies have confirmed that FOXA2 is crucial for inhibiting tumor metastasis by promoting EMT [184,185]. Staphylococcal nuclease domain-containing protein 1 (SND1) is highly expressed in CC tissues and is positively correlated with a shorter survival time [73]. Cell experiments confirmed that SND1 promotes Smurf1 expression in CC cells. Smurf1 interacts with FOXA2 and induces its ubiquitination and proteasome degradation, an essential mechanism by which SND1 promotes metastasis in CC cells [73].

NEDD4L
NEDD4L not only regulates the function of epithelial sodium channels to achieve cell stability and homeostasis but also prevents cancer development by inhibiting the transforming growth factor-β(TGF-β) signaling pathway [186]. TGF-β is a multifunctional polypeptide growth factor that plays a tumor suppressor role in normal and precancerous cells but plays a cancer-promoting role in cancers [187]. Yilmaz et al. were the first to study NEDD4L expression in EC, and their findings showed that NEDD4L expression levels are reduced in EC patients compared with benign endometrial lesions [74]. This finding implied that NEDD4L may play an essential role in the occurrence and development of EC. Therefore, it is necessary to explore new substrates or pathways related to NEDD4L to provide new clues for inhibiting the occurrence and development of EC.

Mutations in E3 Ligases in EC and CC
Mutations are also important causes of cancer. However, E3 ligase mutations in EC and CC have not been systematically studied and summarized. We searched and summarized the status of E3 ligases leading to EC and CC using the cBioPortal database. However, the specific effects of these mutated sites on E3 ligase, such as activation or inactivation, have not been confirmed (Tables 2 and 3).   Considering these mutation sites and mutation types, we found that these E3 ligases are dominated by missense mutations in EC and CC and are mainly found in EC. The discovery of new mechanisms of E3 ligase mutation and the development of EC and CC can provide new therapeutic targets for subsequent treatment. Therefore, other E3 ligases can be similarly explored and further explored in their functions to help find better therapeutic targets.

Treatment
Increasing evidence suggests that E3 ligases are related to the development of EC and CC, two highly lethal gynecological malignancies, at multiple levels. Although this review summarizes and catalogs the E3 ligases involved in EC and CC, the genetic alterations of these components in human diseases have not been fully evaluated. Therefore, identifying therapeutic targets for new drugs is very important for the development of therapeutic strategies.

Targeting Proteasomes for EC and CC Therapy
Bortezomib was the first selective proteasome inhibitor approved for clinical, which has led to the development of many other proteasome inhibitors and has opened up a new approach for treating human malignant tumors by regulating the UPS [188,189]. A previous study showed that bortezomib inhibited EC cell proliferation and proteasome activity and induced G2/M phase arrest and apoptosis [190]. Interestingly, sorafenib can also inhibit EC development [191]. However, sorafenib enhanced the activity of UPS through the AKT/GSK3β and ERK pathways and promoted the degradation of the anti-apoptotic protein Mcl-1. As potential anticancer drugs, HIV protease inhibitors (HIV-PIs), such as lopinavir and indinavir, have been shown to selectively inhibit the 26S proteasome in CC [192,193]. Mechanistically, these protease inhibitors damage the cellular proteasome by targeting chymotrypsin activity at the protein breakdown rate, ultimately preventing the degradation of p53 and increasing the levels of tumor suppressor proteins [194][195][196][197]. Although drugs targeting the 26S proteasome remain at the cellular level for the treatment of EC and CC, they also provide an experimental basis for subsequent clinical applications. Similarly, the development of small-molecule inhibitors (SMIs) that can specifically target E3 ligases, thereby reducing the ubiquitination of its substrate, is currently the most promising therapeutic approach. Notably, owing to the specificity and diversity of E3 ligases, inhibitors targeting E3 ligases have lower cytotoxicity and better therapeutic efficacy than proteasome inhibitors. 6.2. Targeting E3 Ligases for EC and CC Therapy 6.2.1. Patient-Derived Tumor Xenograft (Pdx) E3 ligases have received increasing attention as therapeutic targets in EC and CC cells. However, SMIs of these identified E3 ligases involved in EC and CC have been mainly focused on fundamental research and have not been validated for clinical trials and applications. The currently developed SMIs capable of targeting E3 ligases have shown significant antitumor activity in mouse tumor xenograft models, such as XI-001. The pseudourea derivative XI-001 compounds identified as p53 activators by high-throughput screening were used to kill cancer cells but did not affect normal cells [198,199]. Zhang et al. demonstrated that XI-001 could inhibit the growth of xenograft tumors in HeLa tumor-bearing mice and enhance the cytotoxic activity of cisplatin in vitro and in vivo. Mechanistically, XI-001 inhibited the interaction between MDMX and p53, reduced MDMX levels, and stabilized p53 in a dose-dependent manner. Moreover, MDMX colocalizes with E6AP and appears to be a novel binding partner of E6AP that promotes p53 ubiquitination [102]. TBP-like protein (TLP) is a member of the TBP family and can prevent p53 degradation by disrupting the p53-MDM2 interaction. TLP inhibits MDM2-driven nuclear export of p53 to retain it in the nucleus [80]. In terms of physiological significance, TLP can significantly inhibit the growth of solid tumors of the CC in mice. Thus, TLP establishes a novel mechanism for the activation of long-acting p53 to determine cell fate.

Preclinical
Mouse models can be constructed to confirm the tumor suppressive effect of E3targeting inhibitors, which can be further applied in the clinical stage. However, more studies have focused on fundamental studies of putative targets. In addition, owing to the continuous discovery of the oncogenic role of E3 ligases in EC and CC, the exploration of corresponding specific inhibitors has gradually become a research interest, and some progress has been made, such as targeting E6AP, MDM2, and HUWE1 in CC and targeting SCF Skp2/Cks1 and MDM2 in EC. This section focuses on E3 ligase inhibitors of these potential therapeutic targets and their roles in the treatment of EC and CC (Table 4). Competitively inhibit the binding of SCF Skp2/Cks to p27 and mediate p27 accumulation Preclinical [206] As previously described, the E6 oncoprotein expressed in high-risk HPV can regulate p53 levels and transcriptional activity through the ubiquitination pathway in the presence of E6AP [59,207]. Therefore, the design and development of novel anticancer drugs targeting E6AP-p53 have gradually become a research interest. E6AP inhibitors associated with CC include RITA and quercetin [204,208] (Table 4). A study by Zhao et al. confirmed that certain substances that reactivate p53 and induce apoptosis in tumor cells can promote the formation of a ternary complex between p53 and E6/E6AP to protect p53 from ubiquitination and degradation and induce its accumulation [204]. Flavonoids are widely found in most plants. It is commonly associated with various biological activities, including promoting the production of reactive oxygen species (ROS), repairing DNA damage, and restoring the function of p53 [208,209]. Several studies have shown that quercetin, a polyphenolic flavonoid, has anticancer activity against multiple cancers. For example, quercetin induces apoptosis in melanoma cells via a p53/Bax-dependent mechanism while increasing ROS production [200]. Quercetin arrests the cell cycle in the G2 phase and triggers apoptosis in CC cells. Furthermore, quercetin binding to E6 disturbed the interaction between E6 and E6AP [208]. Ultimately, this process suppresses the degradation of p53.
MDM2 can tightly bind to the p53 trans-activation domain and promote ubiquitindependent proteasomal degradation [210]. Thus, inhibitors targeting MDM2 may restore the function of p53 and tumor sensitivity to radiotherapy and chemotherapy by disrupting the p53-MDM2 interaction [211]. It is encouraging that some inhibitors targeting MDM2, including idasanutlin, Nutlin-3, and MI-21, have entered the clinical stage for the treatment of PCa [31]. However, the efficacy of these inhibitors in EC and CC remains unclear. Natural products are widely used in clinical practice, and their use has exceeded 50% at present. Tulbaghia violacea (TV) and Agave palmeri (AG) are two plant extracts that act as potential apoptotic inducers in CC. Treatment of CC cells with these two plant extracts has been shown to significantly reduce MDM2 expression; however, the exact biological mechanisms are still unclear [202]. In addition, another MDM2-targeting small-molecule compound, CP-31398, a synthetic styrene quinazoline, was able to down-regulate MDM2 expression and restore p53 mutant function in EC [205].
Several other drugs have been found to regulate substrate degradation by E3 ligases. For example, Androg, a common traditional medicinal plant in Asia, disrupts HERC4 and SMURF2 activities to restore p53 function in HPV16-positive CC cells [201]. Tobramycin can be used as a targeted therapy to prevent or treat HUWE1 overexpressing CC [67]. It specifically binds to the HECT domain and inhibits the degradation of MCL1. In addition, tobramycin regulates the G2/M transition and inhibits the proliferation of CC cells in vitro [67]. Cellular inhibitor of apoptosis protein 1 (CIAP1) is highly expressed in CC and up-regulates resistance to radiotherapy and chemotherapy. Bestatin-methyl ester (ME-BS) directly interacts with CIAP1, promotes its RING domain-dependent autoubiquitination, and promotes proteasomal degradation of CIAP1 [212]. The inhibitory effect of ME-BS on CC provides a novel strategy for manipulating the intrinsic ubiquitin ligase activity. Pavlides et al. developed a novel drug-binding approach to identify the activity of multiple SMIs of SCF Skp2/Cks1 E3 ligase (Skp2E3LIs) [206]. Skp2E3LIs stabilize p27 in the nucleus and mediate cell cycle arrest in the G1 phase without cytotoxicity.

Targeting the Substrates of E3 Ligase and Neddylation for EC and CC Therapy
Cullins are the best-characterized neddylation substrates [213]. Currently, advances have been made in exploiting inhibitors for neddylation modifications. An increasing number of studies have confirmed that the small molecule MLN4924 can inactivate CRL function and induce autophagy and apoptosis by blocking the neddylation of Cullin in EC and HCC [214,215]. Some E3 ligases are considered tumor suppressors of EC, and the accumulation of carcinogenic substrates caused by frequent mutations is the cause of cancer. Therefore, the development of corresponding E3 analogs or inhibitors targeting oncogenic substrates may be a neoadjuvant therapy for CC and EC. FBXW7 mutations increase the phosphorylation of proteins, including SRC-3, cyclin E1, c-Myc, rictor, GSK3, AKT, and P70S6, which are essential causes of EC [153]. Urick et al. provided evidence that two drugs can significantly reduce the survivability of EC cells with FBXW7 mutant in vitro: SI-2, an SMI of SRC, selectively targets SRC-3; dinaciclib inhibits the activity of Cdk2 as well as Cdk5, 1, and 9 [153]. In addition, the use of adenoviruses as live vectors encoding p53 has shown progressive results in patients with CC. As mentioned above, CUL3 SPOP inhibits the progression of EC and PCa by degrading oncoproteins [216]. Interestingly, endometrial CUL3 SPOP mutants that sensitize BET inhibitors spontaneously degrade BET proteins; however, PCa-specific CUL3 SPOP mutants impair the degradation of BET protein and promote resistance to its pharmacological inhibition [106]. This difference indicates that mutations in the same E3 ligase domain can have different drug sensitivities depending on the tumor.

PROTACs
Inhibitors targeting EC-and CC-related E3 ligases are still at the cellular level and far from the clinical stage. At the same time, the development of these inhibitors has difficulties, such as drug resistance, side effects, and reduced activity against many proteins. Thus, the emergence of new models of proteolysis-targeting chimeras (PROTACs) that control substrate levels provides extraordinary strategies for enhancing drug development [217]. Instead of inhibiting the function of the protein of interest (POI), PROTACs bind an E3 ligase and intracellular POI and label it with ubiquitin, and then induce cells to remove POI by UPS [218]. Importantly, PROTACs can be recycled to exert stronger biological effects [219]. Crews et al. reported the first PROTAC small molecule with high cellular permeability and biostability that can successfully mediate ubiquitination degradation of androgen receptors (ARs) in CC cells at 10Mm [220]. This PROTAC associates a nonsteroidal derivative of the selective androgen receptor modulators (SARM) with nutlin to recruit MDM2 (Figure 3). Recently, a new generation of PROTACs targeting AR has been developed, including ARV-110, ARD-266, ARD-61, and ARD-69 [65]. Currently, ARV-110 is entering phase II clinical trials for patients with PCa. Triazolodiazepine-based JQ1 is an inhibitor of Bromo-and Extra-terminal (BET) family of proteins [221]. MZ1 contains two linker ligands, one is JQ1, responsible for recruiting BET proteins, and the other is a von Hippel-Lindau (VHL) ligand [222]. In CC, MZ1 can specifically bind and rapidly degrade BET proteins, especially BRD4 [222]. Similarly, replacing the VHL ligand of MZ1 with the RNF4 ligand leads to another PROTAC drug, CCW28-3 [223]. In addition to MDM2, VHL, and RNF4, cereblon (CRBN), inhibitors of apoptosis (IAPs), DDB1, cul4-related factors (DCAF15 and DCAF16), and RNF114 ligands with PROTACs are offering prospects for the exploration and development of new therapeutic agents [224][225][226][227][228]. cellular permeability and biostability that can successfully mediate ubiquitination degradation of androgen receptors (ARs) in CC cells at 10Mm [220]. This PROTAC associates a non-steroidal derivative of the selective androgen receptor modulators (SARM) with nutlin to recruit MDM2 (Figure 3). Recently, a new generation of PROTACs targeting AR has been developed, including ARV-110, ARD-266, ARD-61, and ARD-69 [65]. Currently ARV-110 is entering phase II clinical trials for patients with PCa. Triazolodiazepine-based JQ1 is an inhibitor of Bromo-and Extra-terminal (BET) family of proteins [221]. MZ1 contains two linker ligands, one is JQ1, responsible for recruiting BET proteins, and the other is a von Hippel-Lindau (VHL) ligand [222]. In CC, MZ1 can specifically bind and rapidly degrade BET proteins, especially BRD4 [222]. Similarly, replacing the VHL ligand of MZ1 with the RNF4 ligand leads to another PROTAC drug, CCW28-3 [223]. In addition to MDM2, VHL, and RNF4, cereblon (CRBN), inhibitors of apoptosis (IAPs), DDB1, cul4 related factors (DCAF15 and DCAF16), and RNF114 ligands with PROTACs are offering prospects for the exploration and development of new therapeutic agents [224][225][226][227][228].  Figure 3. PROTAC-mediated degradation of target proteins through the ubiquitin-proteasome system (UPS). Selective androgen receptor modulator (SARM) and nutlin assemble into a complex that hijacks murine double minute 2 (MDM2). SARM with nutlin binds MDM2 and intracellular androgen receptor (AR), labels AR with ubiquitin, and then induces cells to remove AR by proteasomes.

Conclusions and Perspectives
We summarized the relevant mechanisms and mutation sites of these E3 ligases leading to the development and progression of EC and CC. Intervention with these E3 ligases or specific substrates will facilitate subsequent treatment.
Several SMIs and activators, such as bortezomib, Nutlin, and RITA, have been developed to treat various diseases caused by UPS dysfunction. These SMIs have the advantages of low molecular weight, high cell/tissue permeability, high oral bioavailability, and little off-target. In addition, SMIs are more tolerant to target proteins that maintain normal cell function. However, these SMIs are prone to drug tolerance and cytotoxicity due to the need for frequent dosing. An example is bortezomib-induced peripheral neuropathy. The efficacy of these SMIs requires a high affinity for the target protein, so they can hardly completely inhibit the function of their target. Currently, most of the efficacy testing of SMIs is stalled at the cellular level. This is unfavorable for subsequent clinical applications. Therefore, animal experiments, such as the construction of the mouse Pdx model, can be further verified its efficacy. XI-001 and TLP successfully inhibited the growth of CC tissue in xenograft mice, but their toxicities are currently unknown. For SMIs used in the clinic, toxic side effects, specific affinity to target proteins, and drug absorption need to be further detected.
The development of SMIs is the primary strategy for targeted therapy of E3 ligase dysfunction. However, most SMIs developed have poor potency and selectivity. Therefore, understanding the action mechanism of E3 ligases and the toxicity mechanism of SMIs is critical for drug screening and discovery. The innovation of PROTAC technology solves various difficulties in developing targeted E3 ligase inhibitors and opens a new stage in research and new drug development. However, identifying the best ligand capable of designing PROTAC remains a challenge. Although numerous E3 ligases have been identified, only some can be recruited into chimeric small molecules to degrade target proteins, such as FBXW7, MDM2, VHL, and KEAP1. In addition, several novel PROTACs drugs have been successfully designed and applied to the degradation of oncogenic proteins in CC cells, such as MZI and CCW28-3. Whether they can be applied to the degradation of the same oncogenic proteins in EC cells remains unknown. It may be related to the tissue specificity of oncogenic proteins. Whether the E3 ligases associated with EC and CC summarized in this review can act as ligands for PROTAC also remains unknown. This review also facilitates the search for PROTAC ligands for further explorers. Therefore, we have reason to believe that these PROTACs have promise for the treatment of EC and CC in the future.
E3 ligase dysfunction is essential in the development and progression of EC and CC. By focusing on the development of more targeted SMIs or activators of E3 ligases, the therapeutic effect will be significantly improved, which will also meet the diagnosis and treatment requirements of cancer precision medicine.

Conflicts of Interest:
The authors declare that there is no conflict of interest.