Ubiquitination regulates autophagy in cancer: simple modifications, promising targets

Abstract

Autophagy is an important lysosomal degradation process that digests and recycles bio-molecules, protein or lipid aggregates, organelles, and invaded pathogens. Autophagy plays crucial roles in regulation of metabolic and oxidative stress and multiple pathological processes. In cancer, the role of autophagy is dual and paradoxical. Ubiquitination has been identified as a key regulator of autophagy that can influence various steps in the autophagic process, with autophagy-related proteins being targeted for ubiquitination, thus impacting cancer progression and the effectiveness of therapeutic interventions. This review will concentrate on mechanisms underlying autophagy, ubiquitination, and their interactions in cancer, as well as explore the use of drugs that target the ubiquitin-proteasome system (UPS) and ubiquitination process in autophagy as part of cancer therapy.

Introduction

Autophagy is a cellular process that involves the degradation of cellular components, which is triggered in cells during periods of nutrient deprivation or cellular stress [1]. The autophagy process consists of four essential stages: initiation, nucleation, maturation, and degradation [2]. The far-reaching impacts of autophagy on maintaining organismal balance have led to significant research efforts in identifying potential targets for clinical intervention to regulate the autophagic process for the prevention or treatment of diseases in various pathological conditions [3]. Pro- and anti-tumor effects of organelle-targeted autophagy depends on different circumstances. In the onset and early stages of cancer and tumor development, impaired autophagy promotes the progression of cancer. In the later stages of cancer progression, cancer cells typically use autophagy as a way to cope with the intracellular stress associated with their malignant state and difficult microenvironmental conditions, while also promoting cancer metastasis [4, 5].

Different post-translational modifications (PTMs) on autophagy-related components and upstream effectors have been recognized as crucial mechanisms for regulating autophagy, ultimately influencing the activation and suppression of the autophagy process. The ubiquitination process involves the formation of an isopeptide bond between a lysine residue on the substrate protein and the carboxyl group of glycine 76 on ubiquitin. The attachment of ubiquitin to proteins is carried out through a coordinated series of steps involving E1 activating enzymes, E2 conjugating enzymes, and E3 ubiquitin ligases. These enzymes work together to activate, transfer, and attach ubiquitin to specific protein substrates, thereby regulating various cellular processes [6, 7]. The ubiquitination by some E3 ubiquitin ligases mediates the degradation of proteins involved in specific cellular pathways, thereby playing a role in cancer [8, 9]. The application of modulating the ubiquitination, especially the proteasomes to treat cancer has been applied in clinical settings [ 10-12]. Clinical studies about targeting E3-autophagy protein axis in cancer are rare. It is valuable to discover the relation between the cancer aberrant marker of the E3 ligase expression and autophagy protein content and develop associated drugs in clinical therapy. Ubiquitination plays a role in regulating various stages of autophagy (Fig. 1). Furthermore, the autophagic pathway is triggered to compensate for decreased activity in the UPS, enabling cells to diminish the burden of accumulated proteasome-specific substrates. The connection between the ubiquitination and autophagy is established through the sharing of common regulators or substrates. One prominent molecule shared between the two pathways is ubiquitin itself, which serves as a signal to tag target substrates for degradation by the proteasome or to be recognized by adaptor proteins (such as p62) for the recruitment of targets into autophagosomes during selective autophagy [13]. There have been multiple studies investigating the relationship between the ubiquitination and autophagy. Developing the drug combination application targeting the ubiquitination and autophagy in different situations has potential in cancer therapy. E3 ubiquitin ligase, the most specific and ubiquitous enzyme in the ubiquitination pathway, holds great clinical potential for regulating autophagy in cancer by modulating its activity. Here we review how autophagy is regulated by ubiquitination in cancer and the potential therapy targeting ubiquitination to influence autophagy in cancer.

Fig. 1.

Ubiquitination in autophagy. The initiation of autophagy is stimulated by various factors or can be regulated by other upstream molecules, such as AMPK, mTOR, p53, etc. Subsequently, cup-shaped membrane structures elongate, bend, and eventually close through membrane fission to form double-membrane structures called autophagosomes, capturing specific proteins within itself through cargo recognition. These autophagosomes then fuse with lysosomes. Proteins related to autophagy have been found to undergo ubiquitination modification in both the upstream pathways and the four stages of autophagy and play a role in cancer. Ubiquitination can mediate protein-protein interactions and protein degradation

Ubiquitination-mediated proteins to induce autophagy in cancer modifies

mTOR complex

As shown in Fig. 2, under nutrient-rich conditions, the mammalian target of rapamycin complex 1(mTORC1) could inhibit autophagy through phosphorylation [14]. The increased autophagy by disruption of mTOR through ubiquitination is prominent in cancer. Binding with TNF receptor -associated factor 6 (TRAF6), p62 is necessary for the movement of mTORC1 to the lysosome and its subsequent activation increased. Expression of DEAD box protein 5(DDX5) has been found to reduce the p62/TRAF6-mediated lysine 63-linked ubiquitination of the mammalian target of rapamycin (mTOR), resulting in the inhibition of the mTOR signaling pathway. The function of DDX5-induced autophagy by disrupting p62 and TRAF6 ubiquitination impairs hepatocellular carcinoma(HCC) cell growth [15, 16]. mTOR activation triggers the phosphorylation of DEPTOR, a protein that acts as a negative regulator of both mTORC1 and mTORC2. This phosphorylation process leads to the degradation of DEPTOR through ubiquitination mediated by the βTrCP-SCF complex [17, 18]. In human breast cancer, Ribosomal protein S27-like (RPS27L) decreased expression may reduce the β-TrCP level, which results in activating autophagy by inactivating mTORC1 [19]. This indicates that the ubiquitination of mTORC may participate in breast and liver cancer progression. In pancreatic ductal adenocarcinoma (PDAC), SHOC2 is overexpressed which is associated with poor survival rate. SHOC2 is ubiquitinated and degraded by FBWB7, a SCF E3 ligase, which may block the RAS-MAPK pathway and suppress cancer cell growth. SHOC2 also binds to RAPTOR to inactivate mTORC1, thus causing activating autophagy [20]. Whether the effect on autophagy by overexpressing FBWB7 can improve the survival of PDAC needs to be elucidated. Overexpression of circZKSaa in HCC cells promotes the ubiquitination of mTOR mediated by FBXW7. The downstream pathway may involve autophagy, given that autophagy is stimulated in FBXW7-mTOR under prion infection [21, 22]. The E3 ligases negatively relate to cancer, which decide the ubiquitination substrates and are vital to the whole axis related to cancer development.

Fig. 2.

Ubiquitination of mTOR in cancer. It has shown that mTOR is regulated by ubiquitination. Among them, Cul3-KLHL and TRIM21 indirectly target mTOR through ubiquitination -mediated degradation to boost breast cancer growth and inhibit proliferation and metastasis of NSCLC. In breast cancer, the activated autophagy is also seen with decreased mTOR activity caused by decreased RPS27L. SCF-FBWB7 target SHOCK2 which was shown increased expression in PDAC. While Cul1-FBXW7 directly target mTOR to elevate autophagy and breast cancer growth through ubiquitination. Impairing HCC cell growth by inducing autophagy through DDX5 involves disrupting p62 and TRAF6 ubiquitination on mTOR

Interestingly decreased autophagy by regulation of mTOR is also related to cancer. AGO4 stabilizes tripartite motif (TRIM) 21, which ubiquitinates GRP78 (glucose-regulated protein 78), thus leading to K48-linked degradation of GRP78. Subsequently, it contains autophagy by activating the mTOR pathway. The axis may be the mechanism in AGO4 inhibiting the proliferation and metastasis of non-small cell lung cancer (NSCLC) cells [23]. The E3 ubiquitin ligase complex CUL3-KLHL22 promotes K48-linked polyubiquitination and subsequent degradation of DEPDC5, activating mTOR and inhibiting autophagy. Deletion of kelch-like (KLHL) 22 triggers autophagy activation and suppresses breast cancer growth [24]. Enhancing degradation mediated by ubiquitin in the mTOR pathway may shed a light on cancer treatment.

Ubiquitination-mediated protein in cancer to initiation of autophagy

ULK1-Atg13-FIP200-Atg101

Unc-51 like autophagy activating kinase 1 (ULK1) is crucial to the initiation of autophagy and also subjected to PTM. ULK1 regulates a phosphatase network, which is modulated by various phosphatases to mediate autophagosome expansion, maturation, and lysosome fusion [25]. Substantial examples show that ubiquitination regulate autophagy through degradation of ULK1. Depletion of p32 enhances K48-linked polyubiquitination but inhibits K63-linked polyubiquitination of ULK1, resulting in proteasome-mediated degradation of ULK1 [26]. The E3 ligase neural precursor cell-expressed developmentally down-regulated 4–like (NEDD4L) specifically down-regulates ULK1 protein levels by ubiquitinating ULK1 for degradation by the proteasome [27]. Both p32 and NEDD4L function in stopping the excessive autophagy to keep it at appropriate level. Mitochondrial E3 ubiquitin protein ligase 1 (MUL1) facilitates the ubiquitination of ULK1, leading to the degradation of this protein. It is interesting that while MUL1 promotes ULK1 degradation, it also enhances mitophagy [28]. The recruitment of ULK1 for ubiquitination and subsequent proteolysis by KLHL20 is facilitated by its autophosphorylation. Damaged KLHL20 function on autophagy deteriorated diabetes-associated muscle atrophy [29]. In addition to degradation of ULK1 through ubiquitination in normal conditions, ubiquitination could also regulate its function in cancer. NEDD4L inhibited autophagy and mitochondrial metabolism by decreasing cellular ULK1 or ASCT2 levels, thereby potentially inhibiting the proliferation and viability of pancreatic cancer cells [30]. TRIM27 activates STK38L by ubiquitination, thus STK38L phosphorylates ULK1. The whole process facilitates TRIM27 to ubiquitinate and degrade ULK1 through proteasomal. TRIM27 negatively regulates autophagy, which prevents tumor metastasis. Meantime low level of TRIM27 is associated with decreased primary lesion mass. It may involve in other signal pathways, which needs further investigation [31]. For cancer occurrence and metastasis, impaired autophagy inhibits and promotes cancer progression differently. The E3 ligase BTRC-mediated proteasome degradation of Lys48-linked ubiquitination of ULK1 was enhanced by phosphorylation of ULK1 by MAPK1/ERK2-MAPK3/ERK1 kinase. The impaired mitophagy activates the NLRP3 inflammasome and amplifies the release of cytokines, which may account for breast cancer bone metastasis [32].

Mostly, ubiquitination regulates autophagy protein through K63-linkage. TRIM32 acts as a pro-autophagy factor by promoting ULK1 activity through unanchored K63-linked polyubiquitin [33]. The phenomenon is observed under atrophic stimuli, whether the axis regulates other diseases, like tumor genesis, needs subsequent studies. TRIM16 facilitates K63-linked ubiquitination of ULK1, potentially through its association with cullin ubiquitin ligase components as indicated by co-immunoprecipitation. The modification may stabilize the autophagy regulators [34, 35]. AMBRA1 (Activating molecule in Beclin1-regulated autophagy), when dephosphorylated, interacts with the E3 ligase TRAF6 to facilitate the ubiquitination of ULK1 with K63-linked chains, leading to its stabilization, self-association, and functional enhancement [34]. In cancer, the E3 ligases are attributed to control autophagy and affect disease progression. The formation of Homo-dimerization of TRAF6 is stimulated by Grancalcin(GCA)-mediated K63-linked poly-ubiquitination of TRAF6. GCA activates TRAF6 to stimulate the K63-linked ubiquitination of ULK1, thus leading to induce autophagy. The K63-linked ubiquitination of ULK1 also impedes K48-linked ubiquitination of ULK1. TRAF6-mediated promoted autophagy plays an important role in Imatinib-resistant Chronic myeloid leukemia (CML) patients [36]. SPHK1 enhances the advancement and spread of colorectal cancer by controlling autophagy through TRAF6-induced ULK1 ubiquitination [37]. AQP5 brings TRIM21 to the important autophagy protein ULK1, leading to the ubiquitination of ULK1 and the activation of autophagy. This process enhances the stemness of gastric-cancer stem cells [38]. The elevation of TRAF6 and TRIM21 in different types of cancers through the modification of ULK1 could pave the way for novel developments in cancer therapy.

Ubiquitination modification on Atg protein in ULK1 complex has important features in autophagy and cancer. The LUBAC key component RNF31 mediated proper linear ubiquitination of Atg13 stabilizes the protein, preventing its degradation by the proteasome, thus controlling autophagy initiation and maturation [39].Atg13-Atg101 HORMA dimer serves as a critical component in initiating the autophagy process by enabling the ULK1 complex to recruit Atg9A vesicles to initiate autophagy [40]. HUWE1 mediates the ubiquitination of Atg101 with K48-linked ubiquitin chains, leading to the degradation of Atg101 and the inhibition of autophagy. The negatively regulated autophagy pathway potentially hinders the survival of cancer cells or improves the effectiveness of anti-tumor treatments [41] (Fig. 3). The different function of E3 ligase leads to different autophagy activity, which may provide insight into new targets to cure cancer.

Fig. 3.

Ubiquitination of ULK1 complex in cancer. MUL1, depletion of p32, KLHL20 and TRIM27 adding K48-linked ubiquitin leads to ULK1 degradation. While NEDD4L holds pancreatic cancer through K27 and K29 linked chain. TRIM27 and HUWE1 inhibit cancer and tumor metastasis through degrading ULK1 and Atg101 respectively. However, ULK1 degradation by BTRC is stimulated by phosphorylation of ULK1 and promote breast cancer bone metastasis. RNF31 ubiquitinates Atg13 to initiate autophagy. TRAF6 and TRIM16 binding with dephosphorylated AMBRA and Cullin respectively add K63-linked chain as well as TRIM32. AQP5 and SPHK1 as promoter in gastric cancer and CRC lead to ubiquitination of ULK1. In imatinib-resistant CML, elevated GCA contributes to ubiquitination of ULK1

Ubiquitination-mediated proteins in cancer to nucleation

Class III PI3K complex

Beclin1

Beclin1, known as a multifunctional protein, exerts significant crosstalk roles between autophagy and cancer. PTM of Beclin1 regulate autophagy and have been found to be associated with promoting chemotherapy resistance in cancer [42]. Most E3 ligases have a vital role in inhibiting autophagy through modified Beclin1 to degradation. The stability of Beclin1 is regulated by NEDD4 expression, and depletion of the Beclin1-interacting protein VPS34 leads to NEDD4-mediated proteasomal degradation of Beclin1 through lysine-11-linked polyubiquitination [43]. Autophagy-stimulated degradation of Beclin1 and VPS34 is regulated by KLHL20 [29]. SKP2 functions as E3 ligase for K48-linked ubiquitination at the K402 site of Beclin1, leading to its degradation through the proteasome. AKT1 phosphorylates to active SKP2, thus leading to decreased autophagy [44]. RNF216 can effectively inhibit autophagy by facilitating the proteasome-mediated degradation of Beclin1 through K48-linked ubiquitination [45]. In cancer, a high level of CUL3 was detected in samples from both breast cancer and ovarian cancer. The abnormal expression of CUL3 was associated with poor patient prognosis. Autophagy normally plays an protective role in cancer cell proliferation, While in breast cancer, the degradation of Beclin1 is promoted by CUL3-KLHL38 through K48-linked polyubiquitination, which inhibits autophagic flux and facilitates cell proliferation [46]. Meantime, one of the oncogenic mechanisms of MIR516A is the degradation of Beclin1 via the CRL4 complex, resulting in impaired autophagy in bladder cancer cells [47]. It remains unclear whether restored autophagy in such situation can damage cancer cell, which may give a better understanding of utilizing autophagy to cancer therapy.

For non-degradation modification of Beclin1, it functions in normal condition and may contribute to developing new cancer therapy. Ambra1 functions as an E3 ligase to catalyze K63-linked ubiquitination of Beclin1 during starvation-induced autophagy. The ubiquitination of Beclin1 at residue K437 is essential for initiating autophagy, and the ubiquitinated Beclin1 interacts with Vps34 to enhance its activity [48]. TRIM50, an E3 ubiquitin ligase, interacts with Beclin1, facilitating its K63-linked ubiquitination and binding to ULK1, leading to the activation of Beclin1 and the promotion of autophagy. This modification enhances the assembly of Beclin1 with ULK1, ultimately activating autophagy. Another TRIM family protein is also seen in modification of Beclin1. Expression of TRIM16 alongside Beclin1 resulted in the stimulation of K63-linked ubiquitination of Beclin1 [35, 49]. Whether its function is direct or indirect remains to be discussed. CaMKII directly interacts with Beclin1 and phosphorylates its Ser90 site, leading to K63-linked ubiquitination and activating the autophagy pathway. This pathway also induces the autophagic degradation of Id-1/Id-2. Given that Id proteins have high expression in numerous solid tumors, the degradation of Ids may facilitate the differentiation of tumor cells [50]. TRAF6 functions as an E3 ligase to mediate the K63-linked ubiquitination of Beclin1 in regulating NF-κB activation in TLR4-induced autophagy in macrophages. Since the ubiquitination site in Beclin1 targeted by TRAF6 overlaps with the site where Bcl2 binds to Beclin1, this overlap could potentially contribute to increased autophagy levels [51]. The ubiquitination of Beclin1 at K117 by TRAF6 is essential for the differentiation of osteoclasts stimulated by RANKL [52]. The potential association between the absence of Beclin1 modification and the development of bone tumors remains unexplored. Moreover, the ubiquitin-modulated protein Rubicon has been found to play a role in bone tissue disorders. The HECTD1-Rubicon pathway has already been found related to osteoarthritis pathogenesis, in which Rubicon is degraded through HECTD1-mediated ubiquitination by proteasomal [53] (Fig. 4).

Fig. 4.

Ubiquitination of Beclin1 complex in cancer. FBXL20 and KLHL20 function to degrade VPS34. Meantime, KLHL20 degrades Beclin1 which is same with phosphorylated SKP2, RNF216 and KLHL38. KLHL38 and CRL4 contribute to breast and bladder cancer for their role in degradation of Beclin1. NEDD4 adds K48-linked chain to Beclin1, which facilitates its degradation. TRAF6, TRIM50, CaMKII and TRIM16 regulate autophagy through K63-linked ubiquitination. CaMKII could promote autophagy to degrade Id-1 and Id-2 to suppress tumor. HECT ubiquitinates and down-regulates Rubicon to promote autophagy. UVRAG is ubiquitinated by SMURF1 to activate autophagy, which inhibits HCC. Atg14L is regulated by ZBTB16 and MARCH7 through degrative and non-degradative modification. AMBRA1 is a positive-regulator of Beclin1 complex. DDB1 and RNF2 degrade AMBRA1 to inhibit autophagy in which WASH could directly inhibit AMBRA1 E3 ligase activity consequently suppress Beclin1 ubiquitination

AMBRA1-UVRAG

CSNK1A1 phosphorylates Ultra-Violet Radiation Resistance-Associated Gene (UVRAG) at Ser522, leading to enhanced interaction between UVRAG and RUBCN. The ubiquitination of UVRAG by SMURF1 was negatively influenced by this phosphorylation event, which in turn boosts the activity of PIK3C3. Consequently, this facilitates the maturation of autophagosomes and the lysosomal degradation of EGFR. Ultimately, this process reduces EGFR signaling and suppresses HCC cell multiplication and tumor expansion [54]. When UVRAG interacts with AMBRA1 and Atg14, it facilitates autophagy by promoting interactions within the Beclin1 complex [55]. In unstressed conditions, the regulation of autophagy dynamics is controlled by the interplay between AMBRA1 and cullin4 E3 ubiquitin ligases [56]. WASH can enlist RNF2 to attach ubiquitin molecules to AMBRA1 via K48-linkage, marking it for degradation through the proteasome degradation pathway. The degradation of AMBRA1 disrupts the AMBRA1-DDB1-CUL4A E3 ligase complex, thereby inhibiting the ubiquitination of Beclin1. In such a way, it can inhibit autophagy ultimately [48, 57]. The ubiquitination of AMBRA1 could directly control PIK3C3, offering potential implications for connecting with HCC (Fig. 4).

Other proteins in PIK3C3

The generation of PI3P on different membranes, such as endosomes, necessitates the presence of VPS34. The inhibition of autophagy can be led to the suppression of VPS34 [55]. The FBXL20 protein acts as an adaptor for the SCF complex, which regulates the ubiquitination and proteasomal degradation of Vps34 depending on p53 when the DNA damage response is activated [58]. Despite numerous studies demonstrating the regulation of autophagy flux by ubiquitination targeting VPS34 and Atg14L, none have expanded its utility in cancer research advancements. Phosphorylation of several serine/threonine residues in Atg14L by mTOR was identified, and mutating these sites to alanine enhanced both PI3K and autophagy activity [59]. A common downstream mechanism that is modulated by GPCR signaling for controlling autophagy is the ubiquitination and degradation of Atg14L, which is regulated by ZBTB16 [60]. MARCH7-mediated mixed polyubiquitination of Atg14 with K6, K11, and K63 linkages results in the insoluble aggregation of Atg14, ultimately leading to the inhibition of autophagy [61] (Fig. 4). Considering the demonstrated significance of ubiquitination in PIK3C3 for cancer, modifications of VPS34 and Atg14L through ubiquitination might also contribute to cancer advancement.

Ubiquitination-mediated proteins in cancer to elongation

WIPI2

Researchers have identified specific membranes in which WIPI-2 accumulates upon autophagy induction [62]. DFCP1 and WIPI2b interact with PI3P. Subsequently, WIPI2b recruits the Atg12∼Atg5–Atg16L1 complex, facilitating the lipidation of LC3 on the phagophore [63, 64]. During mitosis, activated CRL4s recruit WIPI2 and facilitate its polyubiquitination and subsequent proteasomal degradation, thereby resulting in the inhibition of autophagy during mitosis [65]. In addition to its role in physiology, its presence is also found in diseases. It was reported that high expression of CRL4s was observed in various cancer and its role in tumorigenesis [66–68]. This may cast a light on cancer treatment for the oncogenic function of CRL4s. Besides, phosphorylation of WIPI2 by mTORC1 enables its interaction with the E3 ubiquitin ligase HUWE1, leading to ubiquitination and subsequent proteasomal degradation [69]. Further researches are needed to demonstrate the potential of targeting WIPI2 in cancer therapy using HUWE1.

Atg3

Atg3 is essential not only for the canonical autophagy pathway but also for noncanonical LC3 lipidation, facilitating Atg8/LC3 lipidation in both processes [70]. By targeting Atg3 for proteasome-mediated degradation, HRD1 negatively correlates with autophagic flux in NSCLC cells, thereby promoting NSCLC metastasis through the impediment of autophagic MIEN1 degradation [71] (Fig. 5). Its role in autophagy and cancer makes it a meaningful target used in future therapy.

Fig. 5.

Ubiquitination of two ubiquitin-like ligase system. Atg12 is activated by the E1 enzyme Atg7, forming a thioester bond between Atg12 and Atg7. TRIM32 and TRIM7 could add K63-link chain to boost autophagy. While NEDD4 exerts inverse role to degrade Atg7 and Atg5. Subsequently, after modification by the ubiquitin-like conjugating enzyme Atg10, Atg12 forms a heterodimer with Atg5. Finally, Atg12-Atg5 associates with Atg16 to form a complex. TRIM16 enhances the Atg16 to deal with stimulus, while Gigaxonin degrades it. Pro-LC3 protein is cleaved by the cysteine protease Atg4B, exposing a glycine residue. LC3B-1 could be down-regulated by UBA6-BIRC6. The exposed glycine residue of LC3B-1 is activated by the E1 enzyme Atg7, forming a bond between LC3B and Atg7. Subsequently, LC3B binds to the E2 enzyme Atg3. When Atg3 is degraded by HRD1, autophagy is suppressed and promote NSCLC. Finally, with the action of the E3 enzyme complex Atg12-Atg5, it binds to phosphatidylethanolamine to LC3B-PE. VHL is shown to degrade LC3B-PE. CDC20 contributes to ubiquitination of LC3B-PE and enhanced OSCC and Glioblastoma. LC3B-PE captures CTNNB1 following by its degradation, which inhibits CRC. The whole process is intensified by TRAF6, while in colorectal cancer patients with lymph node metastasis, TRAF6 is down-regulated

Atg5-Atg12-Atg16L1

Atg7 is essential for attaching Atg12 to Atg5 like an E1 enzyme. The process of attaching phosphatidylethanolamine (PE) to LC3 involves Atg3 acting as an E2 enzyme and the Atg5–Atg12 complex acting as an E3 enzyme [72]. TRIM21 directly targets Atg5 and facilitates the ubiquitin-dependent degradation of Atg5 in Multiple myeloma (MM) cells. TRIM21 contributes to bortezomib resistance disruption in MM cells by targeting Atg5 to inhibit autophagy and enhance MM cell death [73] (Fig. 5).

The membrane-binding characteristics of Atg16L1, especially its central membrane-binding domain, which is not necessary for typical autophagy but crucial for the lipidation of LC3B on disrupted endosomes, combined with the potential function of the WD40 domain as a hub for protein binding, enhanced by Atg16L1’s ability to form oligomers, could effectively guide the LC3 lipidation process to specific membranes [74]. A recent report observes that Atg16L1 is ubiquitinated by Gigaxonin-E3 ligase, depletion of Gigaxonin induces a massive aggregation of Atg16L1 and impairs phagophore elongation, which is rescued upon reintroduction of the E3 ligase [75], suggesting that E3 ligase, like Gigaxonin, may be a promising therapeutic target to modulate autophagy activity for various diseases, including cancers. Atg16L1 and TRIM16 collaborate in autophagic responses to various forms of endomembrane damage. Following Leu-Leu-O-Me (LLOMe) treatment, the interaction between TRIM16 and Atg16L1 is strengthened, leading to enhanced K63 ubiquitination of Atg16L1 by TRIM16 [35] (Fig. 5). Although the endomembrane system plays an important role in cancer, it still has to be clarified whether impairment of the interaction contributes to the pathogenesis of the cancers.

Atg7

TRIM family function in regulating Atg7 to control autophagy. Under conditions of reactive oxygen species (ROS) stimulation, TRIM32 was discovered to facilitate the K63-linked ubiquitination of Atg7 to initiate autophagy [76]. The K63-linked ubiquitination of Atg7 was triggered by a wide range of conditions. This ubiquitination was notably enhanced by Atg7, and the RING domain of TRIM7 was necessary for Atg7 ubiquitination. This entire process was essential for Atg7’s role in autophagy and in combating L. monocytogenes infection [77]. Circumsporozoite protein (CSP), when translocated from the parasitophorous vacuole (PV) into the cytoplasm of hepatocytes, can be transported into the nucleus, where it upregulates the transcription of E3 ligase NEDD4. This leads to the ubiquitination of Atg5 and Atg7, ultimately inhibiting the IFN-γ-mediated killing of EEFs [78] (Fig. 5). While the regulation of Atg7 has been identified in resistance to foreign pathogens, its regulatory network in cancer remains to be established.

LC3 and GABARAP

The isolation membrane, facilitated by two ubiquitin-like ATG conjugation pathways, namely the Atg12-Atg5 and Atg8/LC3 conjugation systems, develops into a sealed bilayer membrane structure known as the mature autophagosome, which consists of both inner and outer membranes [79]. CDC20 can directly interact with LC3 and facilitate LC3 ubiquitination and degradation via the proteasome, ultimately resulting in the suppression of autophagy. Knockdown-CDC20 may stabilize LC3 thereby inhibiting cardiac hypertrophy [80]. CDC20 has a pro-survival effect on tumors depending on its ubiquitination modification capacity [81]. Its expression regulates Glioblastoma generation and the high level of it represents a poor prognosis in Oral Squamous Cell Carcinoma (OSCC) [82, 83]. Whether CDC20 modification on LC3 accounts for cancer needs further investigation. Contrary to the presumed negative impact on ubiquitinated LC3 in cancer, Ubiquitination modification on LC3 is significant in preventing cancer progression. TRAF6 binding and catalyzing LC3B with K63-linked polyubiquitination promoted the recognition of CTNNB1 by LC3B for selective autophagic degradation, which could inhibit colorectal cancer (CRC) metastasis. While in CRC patients with lymph node metastasis, TRAF6 underwent ubiquitinated degradation [84]. In renal cell carcinoma (RCC), the loss of function of VHL is common. VHL adds ubiquitin to LC3B, resulting in a decrease in LC3B expression [85]. While other studies have identified modifications of LC3 through ubiquitin, its role in cancer has not been elucidated. UBA6 and BIRC6 mediate monoubiquitination degradation of LC3B by proteasomal (Fig. 5). Depletion of up-stream of LC3B degradation supported clearing protein aggregates, which may contribute to neurodegeneration disorders like Parkinson’s disease (PD) [86].

LC3s and GABARAPs utilize a LIR motif to enlist PLEKHM1 to autophagosomes. PLEKHM1’s interaction with the homotypic fusion and protein sorting (HOPS) complex facilitates the fusion of autophagosomes with lysosomes [87, 88]. The centriolar satellite protein (PCM1)-containing centriolar satellites (CSs) prevent centrosomal GABARAP from Mib1-mediated ubiquitination proteasomal-dependent degradation and aid in delivery to autophagosomes. The process may serve as a storage pool ready for acute autophagy induction [89] (Fig. 6). There is scarce research indicating the role of ubiquitination in GABARAP and its implications in cancer.

Fig. 6.

Ubiquitination in fusion and cargo recognition. Ubiquitinated Vmp1 plays a crucial role in Omegasome formation, essential for autophagy activation, and its activation contributes to resistance to cancer therapy. HUWE1 and CRL4s mediate degradation of WIPI2 to regulate autophagy flux. Prostate cancer inhibits CRL3-SPOP-mediated ubiquitination of p62. XIAP inhibits autophagy through p62 degradation thus inhibiting breast and colon cancer. Moreover, TRIM27, keap/Cul3, NEDD4-1 and RNF166 all shown to promote ubiquitination of p62. As autophagy receptor, OPTN is modified by HACE to increase autophagy suppressed lung cancer cell. In Cs, PCM protect GABARAP from Mib1-mediated degradation and help its transport into autophagosome. During fusion process, TRAF6 ubiquitinate RAB7 promoted interaction of STX17 and RAB7 thus boosting lysosomal and autophagosome fusion. Cul4-DDB-WDFY and SCFFBXO27 act on LAMP2 to inhibit lysophagy

VMP1

VMP1 (vacuolar protein sorting-associated protein 1) is crucial for autophagosome biogenesis in dissociating ER-phagophore [90, 91]. Research has revealed a deficiency in the autophagy process in Dictyostelium cells that do not have VMP1. This protein is essential for the removal of ubiquitinated protein aggregates through autophagy [92]. Recent Studies suggest that targeting VMP1 has potential application value in Neuropathy and cardiac disease [93–95]. VMP1 undergoes ubiquitination in the early steps of autophagosome biogenesis, and it maintains this ubiquitinated state as part of the autophagosome membrane throughout the process of autophagic flux until autophagosome-lysosome fusion results in autolysosome formation [96]. The E2F1-EP300-VMP1 pathway plays an important role in mediating gemcitabine-induced autophagy in pancreatic cancer cells chemo-resistance [97]. Study shows that the expression of VMP1 is essential in hypoxia-inducible factor 1-alpha (HIF-1α) mediated autophagy, which would explain the Photodynamic therapy (PDT) resistance in colon cancer treatment [98]. Elevated levels of VMP1 expression may indicate poorer prognoses for patients with HER2-positive breast cancer, Consistent with the finding that its low expression in colorectal cancer and negative-association with the malignant feature of the cancer [99, 100] (Fig. 6). In summary, the connection between ubiquitinated VMP1 in autophagy and cancer may elucidate a new target in treating cancer resistance. With the development of new technologies such as CRISPR screening, an increasing number of autophagy regulating factors are being discovered, such as PFAS and TMEM41B [101, 102]. Newly identified autophagy factor TMEM41B exhibits similar structures and functions with VMP1. Moreover, overexpression of VMP1 has been shown to restore autophagy, which means ubiquitination might also play a similar role on TMEM41B.

Ubiquitination-mediated autophagosome lysosomal fusion in cancer

LAMP2

Lysosomal-associated membrane protein 2 (LAMP2) plays a key role in chaperone-mediated autophagy (CMA), where single proteins are directly transported into the lysosomal lumen. This process involves the specific recognition of individual proteins by the cytosolic chaperone heat shock protein family A (HSP70) member 8 (HSPA8) and their subsequent translocation across the lysosomal membrane through interaction with LAMP2 [103]. CUL4A E3 ligase complex contributes to tag impaired lysosomal LAMP2 with K48-link ubiquitin as a signal to initiate autophagy clearance. While evidence suggests that it may not be only E3 complex for LAMP2 ubiquitination modification [104]. Evidence shows that damaged lysosomal membrane exposes LAMP2 to recognition by FBXO27 and subsequent ubiquitination by the SCF^FBXO27 complex [105] (Fig. 6). Whether other diseases such as cancer could utilize E3-LAMP2 axis as a pro-survival or restrain cell proliferation is worth studying.

RAB7

Rab7 plays a crucial role in overseeing the maturation of endosomes and autophagosomes, guiding cargo transport along microtubules, and ultimately facilitating the fusion process with lysosomes [106]. The RING domain of TRAF6 was found to interact with Rab7 and facilitate the ubiquitination of Rab7 (Fig. 6), leading to direct regulation of the binding between Rab7 and STX17. This interaction ultimately promoted the formation of autolysosomes in macrophages infected with BCG [107]. While a few E3 ligases and autophagy-associated protein functions have been discovered in proteins involved in autophagic lysosomal fusion, their impact on cancer remains unclear.

Ubiquitination-mediated cargo recognition in cancer

p62

p62 plays a crucial role in delivering ubiquitinated cargoes for degradation through autophagy via its C-terminal UBA domain or LIR domain. Additionally, the PB1 domain of p62 aids in promoting this process. By binding to ubiquitinated proteins, p62 facilitates their degradation through the UPS pathway. This dual function of p62 in targeting proteins for degradation helps maintain cellular homeostasis and ensures proper protein turnover [108]. CRL3-SPOP catalyzes the formation of non-degradative K6-, K27-, and K29-linked polyubiquitin chains on p62 through ubiquitination. In normal conditions, it suppresses autophagy. While in prostate cancer, mutation of SPOP disrupts the modification of p62, thereby promoting autophagy, which may shed light on a novel target for prostate cancer therapy [109]. Endogenous XIAP degraded p62 through its ubiquitin E3 ligase function, consequently inhibiting autophagy associated with p62. The reverse expression of XIAP and p62 was observed in breast and colon cancers [110]. TRIM21 can directly bind to and ubiquitinate p62 at lysine 7 (K7). This modification inhibits the ability of p62 to dimerize and carry out its sequestration function in response to Oxidative Stress [111]. Evidence shows that reduced TRIM21 expression is linked to a worse prognosis and enhanced cell proliferation in cancer [112]. The negative relationship between E3 ligase and cancer shows promising future by targeting E3 ligase to treat cancer. Additionally, multiple ubiquitination modification that boost autophagy through p62 have been identified, primarily associated with immunity, while their potential protective role in cancer progression remains uncertain. In Trim protein family mediated autophagy, they usually participate in innate immunity such as RNF166 ubiquitinate p62 leading to antibacterial autophagy [113]. The Ubiquitination in selective autophagy-eliminating pathogen also comes to the forefront, PcAV (pneumoniae-containing autophagic vesicles) first recruits the E3 ligase NEDD4 depending on the complex of p62 and the autophagy protein LC3. NEDD4’s recruitment may then promote additional recruitment of p62, potentially through NEDD4 adding K63-linked polyubiquitin chains to p62 [114]. Keap1/Cul3 can promote the ubiquitination of p62 at the C-terminal UBA domain and thereby enhance its autophagic activity [115] (Fig. 6).

OPTN

The selective engulfment of damaged mitochondria by autophagosomes depends on the autophagy receptor OPTN and its kinase TBK1. When OPTN translocates to damaged mitochondria, it recruits LC3B, leading to the degradation of the damaged organelles through fusion with lysosomes [116, 117]. The tumor suppressor HACE facilitates the ubiquitination of OPTN and enhances its binding with SQSTM1, leading to an increase in autophagic flux. The whole process inhibits the growth and tumorigenic potential of lung cancer cells [118] (Fig. 6). In cancer, heightened metabolism, with mitochondria playing a crucial role, suggests that developing new ubiquitination axis could impact cancer development.

Cancer treatment

In cancer treatment, numerous single-drug therapies have achieved remarkable success, where several drugs have been discovered to target E3 ubiquitin ligases to modulate autophagy for cancer therapy. Most drugs targeting RING-E3 ubiquitin ligases promote autophagy to regulate cancer treatment, while those targeting HECT E3 ubiquitin ligases often inhibit autophagy. Moreover, drugs targeting the proteasome have already been applied in clinical practice. As research continues and drug resistance emerges, combination therapies have demonstrated their advantages. Targeting the proteasome and autophagy is a common approach, as autophagy has a dual role in cancer. Therefore, combining drugs that either promote or inhibit autophagy with proteasome inhibitors has been found to be effective in cancer treatment. Besides directly targeting autophagy, regulating E3 ubiquitin ligases to influence autophagy and combining with proteasome inhibitors is another promising approach (Fig. 7).

Fig. 7.

Cancer treatment based on ubiquitination and autophagy. Targeted ubiquitination and autophagy in clinical treatment are mainly divided into monotherapy and combination therapy. Monotherapy primarily involves targeting E3 ligases and the proteasome system. In combination therapy, the main strategies include targeting E3 ligases and autophagy, as well as targeting the proteasome system and autophagy.

In cancer monotherapy, targeting ubiquitination affects autophagy

Target role of E3 ligases in autophagy for cancer therapy

Evidences show that E3 have direct link with cancer. Numerous drugs aim at treating cancer through disruption its E3 activity have already been in experiment (Table 1).

Targeting the E3 ligases that ubiquitinate autophagy-associated proteins can promote autophagy and exert therapeutic effects in cancer. A considerable number of RING E3 ubiquitin ligases have been recognized as having therapeutic potential as targets for cancer treatment. It has been evidenced that p53 as a tumor suppressor targeting genes to activate the AMPK signal and inhibit the mTOR signal [119]. p53 may serve as an up-stream of autophagy initiated by Atg protein. PLCE1 activates MDM2-dependent ubiquitination and degradation of p53, with decreasing autophagy observed, it involves tumorigenesis in esophageal squamous cell carcinoma (ESCC) [120]. Autophagy plays a role in anti-tumor effects within cells along this pathway in ESCC. MI-63, an MDM2 inhibitor, boosted the effectiveness of bortezomib and lenalidomide and induced autophagy-linked apoptosis in both p53 wild-type and mutant models of MM [121].MI-63 probably provide insight into ESCC and MM therapy for its function on p53.

TRIM65 knockdown suppressed human cervical cancer growth by restraining its activity as E3 ligase to degrade p53, consequently enhancing autophagy. In addition, a decrease of Bcl2 and stable expression of Beclin1 seen in knockdown TRIM65 may reveal a new mechanism in TRIM-mediated autophagy to influence cancer [122]. Another TRIM family protein was found to regulate at the transcriptional level, thereby enhancing autophagy. Recently, neddylation of TRIM25 enhances the movement of transcription factor EB (TFEB) into the nucleus and boosts the transcriptional level of autophagy by heightening the K63-polyubiquitination of TFEB. Consequently, this mechanism decreases the sensitivity of tumors to PTX [123]. As for regulation of AMPK, reports show that MAGEA-TRIM28 targets AMPK to proteasome-mediated degradation, subsequently reduction of ULK1 phosphorylation activation and protective autophagy in cancer tumorigenesis. While CRL4-DCAF12 downregulated MAGE-A3/6 by ubiquitin-proteasomal-system may provide a promising therapy [124–127]. As far, targeting AMPK for their role in autophagy to treat cancer is still in theory. Phosphorylation of Beclin1, which facilitates the ubiquitination of Beclin1, leading to the autophagic degradation of Id and promoting differentiation of neuroblastoma cells [50]. Spermidine decreases p300’s ability to acetylate TRIM50. Deacetylated TRIM50 promotes k-63 ubiquitination of Beclin1, thus promoting autophagy [49]. Spermidine may be used as a natural inducer of autophagy to improve neuroblastoma care. Although numerous TRIM family proteins have been found to modify autophagy proteins, there is still a scarcity of drugs that target the TRIM-autophagy axis to treat cancer.

A novel pathway targeting CRL has been discovered. Except for its role in proteasome, MLN4924 inhibits the NAE pathway in cells, disrupting CRL substrate turnover and suppressing the growth of human tumor xenografts through NEDD8 pathway inhibition. MLN4924 also found that efficiently triggered autophagy in various human cancer cell lines in a manner that depended on both the dosage and duration of treatment [128, 129]. MLN4924 induces autophagy by inactivating CRLs, leading to the accumulation of DEPTOR, which inhibits mTORC1 and contributes to MLN4924-induced autophagy. MLN4924 suppresses the growth of cancer cells in vitro and in vivo [130, 131]. Since many CRLs have directly function on autophagy machinery, it will be intriguing to found a new mechanism in MLN4924 curing cancer.

Targeting other cullin complexes, such as ZBTB, holds therapeutic potential. Regulating the ubiquitination levels of Atg14L through GPCR ligands can potentially be a pharmacological strategy to activate autophagy in the central nervous system. This approach may help inhibit neural dysfunction caused by the accumulation of misfolded proteins [60]. This pathway may also indicate new therapeutic targets in cancer, as GPCRs have been found to mediate cancer induction and interactions among cancer cells [132, 133].

Other drugs targeting cullin complexes show immense promise for cancer treatment, but it is unclear whether their therapeutic effects are mediated by autophagy. Thalidomide and its IMiD derivatives lenalidomide and pomalidomide target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN. CRBN, a negative regulator of autophagy activation, is implicated in cancer progression by inhibiting TRAF6-induced ubiquitination of Beclin1. The effect of IMiDs has emerged as successful therapies for MM and 5q-dysplasia [134, 135]. A new type of CRBN E3 ubiquitin ligase modulator, Mezigdomide, has demonstrated excellent effectiveness in patients with MM [136].

SCF-E3 complex could be positive regulators of mTOR, a critical component in cancer therapy. Silencing of ROC1 triggered autophagy by inhibiting the activity of mTOR through the accumulation of the mTOR-inhibitory protein DEPTOR, and also sensitized cancer cells to apoptosis [137]. Elevated DEPTOR levels inhibited mTOR kinase activity by reducing βTrCP1, leading to increased sensitivity of pituitary adenoma (PA) cells to cabergoline. DEPTOR also promoted autophagy-mediated cell death, further enhancing the cells’ responsiveness to cabergoline [138]. Increasing the levels of SCF-βTrCP alone can potentially inhibit autophagy by activating mTOR, or in application with autophagy inhibitors, may serve as a potential therapeutic approach in breast cancer [19]. The regulation of SCF-E3 ligase by F-box protein BTRC has been applied to clinical therapy. The FDA-approved drug trametinib, which is a targeted inhibitor of the MAP2K/MEK-MAPK1/3 pathway, has been found to effectively inhibit breast cancer bone metastasis for reduced ubiquitination level of ULK1 and restored autophagy [32].

In addition to their role in SCF complexes, Skp1 and Skp2 have emerged as promising targets for cancer treatment, offering new avenues for therapeutic intervention. Gartanin in the diet may have a new potential use as a NEDDylation inhibitor, targeting the selective degradation of the oncogene Skp2 and the up-regulation of the tumor suppressor FBXW2. This could lead to the induction of autophagy and the inhibition of growth in prostate cancer cells [139]. 6-OAP treatment effectively inhibits Skp1 E3 ligase activity by competitively dissociating Skp2, NIPA, and β-TRCP from Skp1, showing efficacy in lung cancer [140]. While it functions through arresting mitosis, whether it can act on the autophagy pathway or impact cancer via the autophagy pathway remains unknown. SMIP004, a small molecule inhibitor of Skp2, efficiently stabilizes Beclin1 against proteasome degradation. Its efficiency in cancer therapy still needs further confirmation [44]. The anti-cancer potential of targeting Skp demonstrates promise for future drug development.

There are also some discoveries of targeting other RING E3 ligases to promote autophagy in cancer. Inhibiting the interaction between STAMBPL1 and Sestrin2 thereby inactivating mTORC1 using a cell-permeable peptide could be a potential treatment approach for gastric and colorectal cancers [141]. The suppressive effect of this may lead to the upregulation of RNF167-mediated ubiquitination, thereby triggering autophagy activation, which could be one of its anti-cancer mechanisms. Accelerating autophagy could be conducive to treat cancer. ICCB-19 and Apt-1 facilitate autophagy by mediating K63-linked ubiquitination of Beclin1 through cIAP1/2 and TRAF2. Blocking apoptosis and restoring cellular homeostasis is achieved by inhibiting TRADD with ICCB-19 or Apt-1, thus activating autophagy in cells [142]. Reduced expression of the ubiquitin E3 ligase ASB3 resulted in increased mitochondrial apoptosis and autophagy in HCC through the caspase-8 mediated cleavage of Beclin1 [143]. Therefore, interfering with the activity of ASB3 in HCC may also be a viable approach for developing an effective strategy.

Disturbance RING-E3 ligases can regulate reduced levels of autophagy to advance cancer therapy, underscoring the importance of exploring this mechanism further in cellular studies. Disruption the MARCH5-dependent pathway for p53 degradation could represent a promising approach for the prevention and treatment of HCC [144]. Beyond regulated autophagy-associated proteins, ubiquitination modifications of autophagy receptor molecules have also been found to have therapeutic potential for cancer treatment. CC-885, a thalidomide analog, rapidly degrades BNIP3L, leading to the repression of BNIP3L-dependent mitophagy and increased sensitivity of Acute myeloid leukemia (AML) cells to mitochondria-targeting drugs [145].

Notably, NEDD4 and ITCH, both targeting HECT E3 ubiquitin ligases, exhibit opposing effects on autophagy during the ubiquitination process. Autophagy may have a pro-survival effect on cancer cells. Clomipramine inhibits autophagy by specifically blocking ITCH auto-ubiquitination and p73 ubiquitination thereby disrupting autophagolysosomal fluxes [146]. In the molecular mechanism of small molecule compound 7695-0983 alleviating sorafenib resistance in HCC cells, autophagy is mediated by the downregulation of NEDD4 [147].

Target role of proteasomes in autophagy for cancer therapy

It is becoming more and more clear that interfering with proteolytic pathways is a promising approach for destroying cancer cells. The tumor cells have high proliferative features and high protein turnover. In this case, targeting the UPS would be a promising therapy. For example, Bortezomib has been proven to be a clinical anti-cancer drugs and may have potential in many cancers [10–12]. Bortezomib, induced cytotoxic autophagy in FLT3-ITD mutated AML cells, also showed its ability in treating MM [149].

However, more often than not, autophagy plays a protective role, which is enhanced after the application of proteasome inhibitors. Bortezomib triggers a protective autophagy response in pancreatic and colorectal cancer cells by activating AMPK-ULK1 signaling pathways [150]. Affecting autophagy with Bortezomib may also due to the up-regulation of SESN2 expression resulting in mTOR inhibition [151]. Another mechanism points out that the up-regulation of autophagy by bortezomib stimulus is likely to activate of JNK enzymes and subsequently disrupt the inhibitory interactions of Bcl-2 with Beclin1 [152]. The administration of bortezomib resulted in elevated levels of intracellular ROS, leading to the induction of autophagy and apoptosis. Interestingly, autophagy was found to confer protection against bortezomib-induced ROS production [153]. However, research also observed that Bortezomib disrupted the autophagic flux by blocking p62 degradation, rather than affecting the fusion of the autophagosome and lysosome [154]. Proteasome inhibitors promote EBNA3C polyubiquitinated and autophagic degradation, which may provide a therapy for multiple EBV-associated B-cell lymphomas [155]. Further research may explore its feasibility in vivo. Under nutrient-rich conditions, cells can survive proteasome inhibition if the autophagic machinery can meet the heightened energy demand and degrade protein cargo effectively. Given that isoginkgetin may inhibit proteasome to burden the lysosomes/autophagic machinery and lead to cancer cell death, combining caloric restriction shows significant promise. Additionally, using isoginkgetin in specific circumstance could enhance therapeutic approaches in MM [156]. Petrosaspongiolide M is an intriguing molecule due to its ability to potentially regulate intracellular proteolysis by simultaneously inhibiting the immunoproteasome and autophagy [157]. Whether it can be applied in treating cancer or any other diseases needs further illustration. In general utilizing proteasome inhibitors and its correlation with autophagy fluctuation could confer a novel therapy in cancer treatment.

In cancer combinational therapy, targeting ubiquitination affects autophagy

Combinational therapy of targeting role of E3 ligases and autophagy in cancer

High expression of certain E3 ubiquitin ligases facilitates cancer treatment. Knocking down TRIM21 in MM cells increased their resistance to bortezomib, while overexpressing TRIM21 resulted in heightened sensitivity to bortezomib. When TRIM21 expression is low, combining autophagy inhibitor 3-methyladenine may have the potential to enhance the effectiveness of bortezomib in MM treatment [73]. Autophagy exhibits a tumor-promoting function in the TRIM21 pathway, whereas it has a tumor-suppressive effect in the other pathway. NEDD4L, a ubiquitin E3 ligase, has been demonstrated to promote autophagy and inhibit proteasomal activity in MM cells. This finding reveals a promising new therapeutic avenue for MM treatment, where autophagy-inducing agents, rapamycin, could be used in NEDD4L-deficient MM or NEDD4L activators could be employed to enhance the efficacy of treatment [158]. Furthermore, targeting UBR5-mediated autophagic degradation of AGR2 or combining autophagy activators such as rapamycin with cancer therapy may help overcome resistance to combination treatment regimens [159]. In addition, some drugs have been shown to inhibit E3 ubiquitin ligase function as part of combination treatment strategies. Compound A (CpdA) has been shown to inhibit the function of the SCF Skp2 E3 ubiquitin ligase complex, leading to cell death through the activation of autophagy in MM cells. This finding suggests that co-targeting autophagy and cell death pathways may be an effective therapeutic approach [148]. The elevation of autophagy induced by MI-63 may contribute to the apoptosis of tumor cells, suggesting that combining MDM2 inhibitors with autophagy activators could provide a promising strategy to overcome resistance in MM therapy [121]. These findings imply that distinct ubiquitin ligases can have substrate-dependent effects, resulting in autophagy exerting contradictory roles in MM. The concomitant use of autophagy regulators and E3 ligase inhibitors holds great promise as a therapeutic approach.

Combinational therapy of autophagy inhibitors and the proteasome inhibitors in Cancer

Elevated autophagy has reverse effectiveness with proteasome inhibitors when treating cancer. Reports found that Bortezomib-induced autophagy leads to a relative drug resistance in diffuse large B-cell lymphoma (DLBCL) cells by removing I-κBα [160]. Cell survival in gynecologic cancer cells resistant to proteasome inhibitors and histone deacetylases (HDAC) inhibitors through autophagy-mediated mechanisms [161]. Drugs developed to inhibit both the UPS and autophagy pathways show promise in cancer treatment. PI1840, noncovalent proteasome inhibitors, attenuated the migration and invasion capabilities of the osteosarcoma (OS) cells. Furthermore, when PI-1840-induced autophagy was blocked, an increase in the survival rate of the OS cells was observed [162].

Mostly, for autophagy has pro-survival in cancer therapy, autophagy inhibitor’s potential was tested in cancer therapy consistent with proteasome inhibitors (Table 2). MLN2238 inhibited cellular proliferation, arrested the cell cycle at the G2/M phase, facilitated apoptosis, and triggered cytoprotective autophagy in intrahepatic cholangiocarcinoma (iCCA) cells [163]. Knocking down Class III phosphatidylinositol-3 kinase Vps34 or Atg5/7 to inhibit autophagy heightened the sensitivity of gastric cancer cells to the growth-inhibiting effects of MG-132 [164]. Verteporfin combined with MG-132 can be used to trigger the regulation of autophagy and protein homeostasis, offering a promising therapeutic approach for treating OS [165]. It was also reported that the use of bortezomib in combination with 3-MA or Atg7 siRNA could offer new possibilities for treating glioblastoma [166]. Clinical trials have shown that combination therapy with hydroxychloroquine (HCQ) is effective in treating various types of cancer [167–169]. The combined use of proteasome inhibitors and autophagy inhibitors is potential in treating multiple cancers.

Reducing autophagy could potentially enhance the effectiveness of proteasome inhibitors in cancer treatment. Chloroquine (CQ), a lysosome inhibitor, could potentially enhance the effectiveness of bortezomib-based treatments for Mantle cell lymphoma by blocking the autophagic breakdown of NOXA [170]. The use of carfilzomib in combination with chloroquine proved to be highly effective in treating MM [171]. Chloroquine also alleviated the cytotoxicity of carfilzomib in acute promyelocytic leukemia (APL) cells [172]. Simultaneous blocking of autophagy and the proteasome work together to effectively trigger cell death in MM [173]. Meanwhile, in clinical trials, the effectiveness of the proteasome inhibitor bortezomib in myeloma therapy is enhanced by inhibiting autophagy with hydroxychloroquine, leading to increased anti-myeloma efficacy [174]. The irreversible inhibition of proteasomes could induce cellular stress, which synergizes with the accumulation of damaged cellular components that should be cleared by autophagy. Therefore, the combination of HCQ and carfilzomib may improve the treatment of myeloma patients [175]. Bafilomycin A and verteporfin, which are Atg inhibitors, enhanced apoptosis in FLT3-ITD-driven leukemic cells induced by bortezomib [176]. Enhancing their anti-tumor effects could potentially be achieved by co-treating with an autophagy inhibitor, which may hinder pro-survival autophagy and thereby augment the antitumor impact of the combination of the STAT3 inhibitor Napabucasin and MG-132, likely through the induction of apoptosis via a mitochondria-dependent pathway in a significantly synergistic anti-proliferative manner [177]. Furthermore, research has uncovered other avenues of autophagy inhibitors that play a crucial role in cancer therapy.

Inhibitors, such as 3-bromopyruvate and Metformin related to glucose metabolism can also modulate autophagy, potentially offering benefits for cancer therapy. The Autophagy induced by HK2 is “oncogenic” in MM, especially in myeloma cells within the hypoxic bone marrow environment. The combined anti-myeloma effects of proteasome inhibitors in normoxia conditions and HK2 inhibitor 3‐bromopyruvate in hypoxic conditions may have resulted in complementary therapeutic effects against myeloma cells [178]. Treatment of myeloma cells with bortezomib resulted in elevated GRP78 levels and the activation of GRP78-dependent autophagy. Inhibiting the functional activity of GRP78 disrupted autophagy and enhanced the anti-myeloma effect of bortezomib [179]. Metformin inhibits GRP78, a critical factor in promoting bortezomib-induced autophagy, and increases the effectiveness of bortezomib in treating myeloma [180]. The combination of ONX0912 and autophagy inhibitors has great potential, as blocking autophagy can increase apoptosis of HCC cells. ONX0912 inhibits cell growth and enhances of mitophagy, which has potential in liver cancer drug application [181].

Combinational therapy of autophagy enhancers and the proteasome inhibitors in cancer

Intriguing, proteasome inhibitors induced autophagy are also observed to help cure cancer. Proteasome inhibitors have the potential to treat VHL-deficient RCC for VHL targeting LC3B which leads to excessive autophagic cell death [85]. The expression of AGR2 is notably reduced by the proteasome inhibitors MG-132/bortezomib at both the mRNA and protein levels in lung cancer cells. MG-132 aids in the degradation of K48-linked polyubiquitinated AGR2 by the E3 ligase UBR5 through the activation of autophagy [159]. AGR2 could disrupt efficiency in cancer, whether joint application of bevacizumab and bortezomib may improve the efficiency of cancer therapy needs further investigation and clinical trial [188]. MLN9708 exhibits antitumor properties in breast cancer and can enhance the sensitivity of breast cancer cells to Dox therapy [189]. With the emergence of drug resistance or diminishing therapeutic efficacy, there is a growing focus on combined drug therapy despite the established use of proteasome inhibitors in clinical settings.

Promoting autophagy may act as synergy with proteasome inhibitors in cancer therapy. This might be associated with the distinct roles of autophagy in various cancers. Targeting G9a/GLP induces autophagy-associated apoptosis by inactivating the mTOR/4EBP1 pathway and decreasing c-MYC levels. When combined with bortezomib, G9a/GLP inhibitors BIX01294 led to a greater decrease in tumor burden and significantly extended survival in MM [190]. Bortezomib/romidepsin also effectively triggered apoptosis and autophagy in gastric carcinoma (GC) by stimulating MAPK- and ROS-dependent autophagy as well as caspase-dependent apoptosis [182]. MIR145-3p induces autophagic flux in MM cells by directly targeting HDAC4, resulting in increased apoptosis [183]. The combined treatment of the proteasome inhibitor MG-132 and AZD6244 synergistically inhibits tumor growth in mice due to blocking the proteasome promotes the autophagic degradation of PDPK1, which physically disrupts the negative feedback signals to the downstream kinase AKT [187]. Bortezomib and temozolomide showed enhanced effectiveness when used together, as bortezomib reduced cell growth, increased DNA damage, and promoted cell death in an Atg5-dependent way, making temozolomide more effective [184]. Temozolomide has the potential to trigger autophagy, subsequently leading to the facilitation of epithelial-mesenchymal transition (EMT) and the promotion of cell invasion and migration. This process may be attributed to the upregulation of the E3 ubiquitin ligase HERC3, which in turn promotes the ubiquitin-mediated autophagic degradation of SMAD7 and activates the TGFβ signaling pathway [185]. The use of solamargine (SM) has been found to increase the expression of genes related to cell death and autophagy. Additionally, a synergistic effect has been observed when SM is used in combination with bortezomib. This pro-autophagy effect enhances the effectiveness of treating MM [186]. The precise mechanism by which SM enhances autophagy for MM treatment is yet to be fully understood. The enhancement of autophagy in cancer therapy primarily involves the degradation of specific key components that influence tumor pathways and autophagy-mediated apoptosis induction, thereby enhancing treatment effectiveness.

Table 1.

Targeting E3 ligases-autophagy for cancer therapy

Drugs	The E3 ligase	Mechanism in autophagy	Cancer	Reference
MI-63	MDM2	MDM2-p53	Myeloma	ADDIN EN.CITE (121)
Mezigdomide	CRBN	/	Multiple myeloma	ADDIN EN.CITE (136)
MLN4924	CRLs	NAE-CRL-DEPTOR-mTOR	Cancer	ADDIN EN.CITE (128-131)
Thalidomide lenalidomide pomalidomide	CRL4-CRBN	CRL4-CRBN- TRAF6- Beclin1	Multiple myeloma 5q-dysplasia	ADDIN EN.CITE (134)
Gartanin	Skp2/FBXW2	NAE-Skp2	Prostate cancer	ADDIN EN.CITE (139)
6-OAP	Skp1	Skp1-Skp2/NIPA/βTRCP-SCF	Lung cancer	ADDIN EN.CITE (140)
CC-885	CRL4-CRBN	CRL4-CRBN-BNIP3L	Acute myeloid leukemia	ADDIN EN.CITE (145)
Clomipramine	ITCH	ITCH/p73	Cancer	ADDIN EN.CITE (146)
7695-0983	NEDD4	FAT10- NEDD4-PTEN/AKT	Hepatocellular carcinoma	ADDIN EN.CITE (147)
Compound A	Skp2	Skp2-SCF- p27	Multiple myeloma	ADDIN EN.CITE (148)

Table 2.

Joint application strategy target proteasome and autophagy in cancer

Proteasome inhibitors	Drugs regulate autophagy	Effect on autophagy	Cancer	Reference
Bortezomib	Chloroquine	Inhibit NOXA autophagic degradation	Mantle cell lymphoma	ADDIN EN.CITE (170)
Carfilzomib	Chloroquine	Inhibit autophagy to promote induced apoptosis	Multiple myeloma, Acute promyelocytic leukemia	ADDIN EN.CITE (171, 172)
Bortezomib	Hydroxychloroquine	Inhibit cytoprotective autophagy	Multiple myeloma	ADDIN EN.CITE (174)
Carfilzomib	Hydroxychloroquine	Inhibit autophagy to promote induced apoptosis	Multiple myeloma	ADDIN EN.CITE (175)
Bortezomib	Bafilomycin A	Inhibit protein hydrolysis promote apoptosis	Acute myeloid leukemia	ADDIN EN.CITE (176)
Bortezomib	Verteporfin	Inhibit protein hydrolysis promote apoptosis	Acute myeloid leukemia	ADDIN EN.CITE (176)
MG-132	Napabucasin	Mitochondrial-dependent apoptosis induction	Ovarian cancer	ADDIN EN.CITE (177)
Bortezomib	3-bromopyruvate	Inhibit HK2 and cytoprotective autophagy	Multiple myeloma	ADDIN EN.CITE (178)
Bortezomib	Metformin	Inhibit GRP78 and cytoprotective autophagy	Multiple myeloma	ADDIN EN.CITE (179)
Bortezomib	Romidepsin	MAPK and ROS dependent autophagic death and caspase-dependent apoptosis	Gastric carcinoma	ADDIN EN.CITE (182)
Bortezomib	MIR145-3p	Inhibit HDAC, up-regulate BCL2L11, inactivate mTOR, promote autophagic death	Multiple myeloma	ADDIN EN.CITE (183)
Bortezomib	Temozolomide	Abrogation of increased autophagy through an Atg5-dependent pathway	Glioblastoma	ADDIN EN.CITE (184, 185)
Bortezomib	Solamargine	Promote autophagy to induce apoptosis	Multiple myeloma	ADDIN EN.CITE (186)
MG-132	AZD6244	Disrupt AKT negative feedback via PDPK1 autophagic degradation	Tumor	ADDIN EN.CITE (187)

Conclusion

Studying upstream pathways and non-autophagy-specific proteins related to autophagy can lead to the development of new drugs. Ubiquitination modification is an important mechanism for regulating autophagy. However, the impact of ubiquitination-mediated modifications of these proteins on autophagy as a therapeutic target in cancer is less explored. In addition to affecting the upstream pathways of autophagy, ubiquitination also acts on the initiation of autophagy, recruitment of proteins, elongation and fusion, and recognition of cargo.

Currently, ubiquitination of autophagy-related proteins mainly facilitates protein-protein interactions or promotes ubiquitination degradation of related proteins. In addition to directly acting on proteins, ubiquitination modification can also regulate autophagy in cancer by influencing the expression of relevant target genes.

Many drugs indirectly regulate the ubiquitination modification of autophagy-related proteins, and these drugs have great potential in the treatment of tumors.

E3 ubiquitin ligases determine the specificity of ubiquitination, making drugs targeting E3 ubiquitin ligases potentially valuable in cancer treatment. However, there is limited research on affecting cancer progression by influencing the E3 ligase-autophagy pathway. Although extensive pharmacological studies have shown that treating cancer by influencing autophagy is possible, it is still necessary to further clarify how drugs specifically target autophagy or which axis is involved in the therapy, and whether drug function through direct or indirect mechanisms.

Most ubiquitination modifications of autophagy proteins mediate the proteasomal degradation of autophagy-related proteins, leading to autophagy inhibition. This type of ubiquitination degradation can inhibit excessive autophagy in physiological conditions, as well as potentially contribute to disease progression when discovered in diseases. Since inhibiting proteasomal degradation of proteins often results in increased autophagy, proteasome inhibitors have been used in clinical myeloma therapy. However, the drug resistant phenomena have been found, so developing new drugs or combining other drugs is imminent. There have been found that combining proteasome inhibitors with autophagy-targeting drugs or using them separately can improve cancer treatment. Bortezomib is now commonly used in clinical trials and is a common drug for treating multiple myeloma. The application of Bortezomib increases autophagy through various mechanisms, and increased autophagy is often considered a protective mechanism of multiple myeloma. Therefore, the combination of autophagy inhibitors can significantly improve the efficacy of treatment.

In cancer therapy, autophagy inhibition is commonly employed as autophagy often acts as a disadvantageous factor in the cancer microenvironment. However, some drugs promote autophagy to induce cancer cell death through autophagy-related apoptosis. Targeting ubiquitination of protein-protein interactions also shows potential as a therapeutic target for tumors.

The role of ubiquitination-modified autophagy-related proteins in cancer and the impact of targeting ubiquitination modification and autophagy is crucial to develop new cancer treatment. Autophagy is often dysregulated in neurodegenerative diseases, heart-related diseases, cancers, and other conditions. Ubiquitination participates in the overall autophagy process and regulates autophagic components, playing a significant role in improving drug resistance and enhancing cancer treatment outcomes. Its application in cancer holds promising prospects.

Abbreviations

APL

Acute promyelocytic leukemia

AMBRA1

Activating molecule in Beclin1-regulated autophagy

CMA

Chaperone-mediated autophagy

CML

Chronic myeloid leukemia

CpdA

Compound A

CRC

Colorectal cancer

CSP

Circumsporozoite protein

CSs

Centriolar satellites

DDX5

DEAD box protein 5

DLBCL

Diffuse large B-cell lymphoma

ESCC

Esophageal squamous cell carcinoma

EMT

Epithelial-mesenchymal transition

GCA

Grancalcin

GRP78

Glucose-regulated protein 78

HCC

Hepatocellular carcinoma

HCQ

Hydroxychloroquine

HDAC

Histone deacetylase

HIF-1α

Hypoxia-inducible factor 1-alpha

HOPS

Homotypic fusion and protein sorting

HSP70

Heat shock protein family A

HSP8

Heat shock protein family A member 8

iCCA

Intrahepatic cholangiocarcinoma

KLHL

Kelch-like

LAMP2

Lysosomal-associated membrane protein 2

LLOMe

Leu-Leu-O-Me

MetO

Methionine oxidation

MFN

Mitofusins

MM

Multiple myeloma

MsrB2

Methionine sulfoxide reductase B2

mTORC1

Mammalian target of rapamycin complex 1

MUL1

Mitochondrial E3 ubiquitin protein ligase 1

NEDD4L

Neural precursor cell-expressed developmentally down-regulated 4–like

NSCLC

Non-small cell lung cancer

OS

Osteosarcoma

OSCC

Oral Squamous Cell Carcinoma

PA

Pituitary adenoma

PcAV

Pneumoniae-containing autophagic vesicles

PCM1

The centriolar satellite protein

PD

Parkinson’s disease

PDAC

Pancreatic ductal adenocarcinoma

PDT

Photodynamic therapy

PE

Phosphatidylethanolamine

PTMs

Post-translational modifications

PV

Parasitophorous vacuole

RCC

Renal cell carcinoma

ROS

Reactive oxygen species

RPS27L

Ribosomal protein S27-like

SM

Solamargine

TRAF6

TNF receptor -associated factor 6

ULK1

Unc-51 like autophagy activating kinase 1

UPS

Ubiquitin-proteasome system

UVRAG

Ultra-Violet Radiation Resistance-Associated Gene

VMP1

Vacuolar protein sorting-associated protein 1

Author contributions

AS and QL conceived the idea and reviewed the manuscript; YW searched the literature and drafted the manuscript; YC and XT corrected the article; GS assisted the manuscript preparation. All authors reviewed and approved the final manuscript.

Funding

This work was supported by the National Natural Science Youth Foundation of China (No. 82002581), National Natural Science Foundation of China (No. 81871888 and 82172942), China Postdoctoral Science Foundation (No. 2020M671375), Jiangsu Province Postdoctoral Research Funding Scheme (No.2020Z261).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.