The role of ubiquitination and deubiquitination in cancer metabolism

Metabolic reprogramming, including enhanced biosynthesis of macromolecules, altered energy metabolism, and maintenance of redox homeostasis, is considered a hallmark of cancer, sustaining cancer cell growth. Multiple signaling pathways, transcription factors and metabolic enzymes participate in the modulation of cancer metabolism and thus, metabolic reprogramming is a highly complex process. Recent studies have observed that ubiquitination and deubiquitination are involved in the regulation of metabolic reprogramming in cancer cells. As one of the most important type of post-translational modifications, ubiquitination is a multistep enzymatic process, involved in diverse cellular biological activities. Dysregulation of ubiquitination and deubiquitination contributes to various disease, including cancer. Here, we discuss the role of ubiquitination and deubiquitination in the regulation of cancer metabolism, which is aimed at highlighting the importance of this post-translational modification in metabolic reprogramming and supporting the development of new therapeutic approaches for cancer treatment.


Background
Metabolic pathways are of vital importance in proliferating cells to meet their demands of various macromolecules and energy [1]. Compared with normal cells, cancer cells own malignant properties, such as increased proliferation rate, and reside in environments short of oxygen and nutrient. Correspondingly, metabolic activities are altered in cancer cells to support their malignant biological behaviors and to adapt to stressful conditions, such as nutrient limitation and hypoxia [1]. Cancer metabolism is an old field of research. Warburg effect observed in the 1920s provides a classical example of metabolic reprogramming in cancer [2]. In the past few decades, enhanced biosynthesis of macromolecules, altered energy metabolism, and maintenance of redox homeostasis have been observed to be essential features of cancer metabolism. Altered metabolism in cancer cells have aroused increasing attention and interest [3]. Because of the generality of metabolic alterations in cancer cells, metabolic reprogramming is thought as hallmark of cancer, providing basis for tumor diagnosis and treatment [1]. For instance, the application of 18Fdeoxyglucose positron emission tomography is based on tumor cells' characteristic of increased glucose consumption [4]. Inhibition of some metabolic enzymes, such as L-lactate dehydrogenase A chain (LDH-A), have been observed to regress established tumors [5,6]. Therefore, research of metabolic reprogramming is of critical importance, which might provide new opportunities for cancer diagnosis and treatment.
Amongst multiple post-translational modification, protein ubiquitination is a common and important process in cells [7,8]. Ubiquitination and deubiquitination have been observed to be dysregulated in various types of cancers. Genetic and epigenetic aberrations, such as mutation, amplification and deletion, can be the common causes of dysregulated ubiquitination and deubiquitination in cancer cells [9]. Ubiquitination and deubiquitination can also be abnormally regulated by transcriptional, translational or posttranslational mechanisms in cancer cells, exerting oncogenic or anti-cancer roles in carcinogenesis [7,8,10]. In recent years, the involvement of ubiquitination and deubiquitination in the regulation of metabolic reprogramming in cancer cells has received a growing body of attention [11]. Given the complexity and importance of both cancer metabolism and protein ubiquitination, the exact roles of protein ubiquitination and deubiquitination in metabolic reprogramming are worth further studies and analyses. The present review will highlight the ubiquitination and deubiquitination system as a regulator of cancer metabolism and discuss future directions focusing on the strategies to improve cancer therapy.

Cancer metabolism
To satisfy nutrient and energy requirements for cells' survival and growth, metabolic pathways are altered in cancer cells, which is called metabolic reprogramming [1]. Metabolic reprogramming is a highly regulated process [12]. Aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1) is one of the most common alterations in proliferating cancer cells, playing a key role in metabolic reprogramming [12]. Under the stimulation of amino acids, mTORC1 can be activated, which subsequently exerts various biological effects by activation of different downstream targets, such as hypoxia-inducible factor 1 (HIF-1) and sterol regulatory element-binding protein (SREBP) [12,13]. Proliferating cancer cells require elevated synthesis of protein, lipid and nucleotide. Glycolysis can be upregulated by mTORC1 activation, providing more glycolytic intermediates for biosynthesis of these macromolecules [14]. Moreover, mTORC1 activation promotes glutamine uptake to maintain mitochondrial ATP production [15]. Fatty acids can also supply carbon to the tricarboxylic acid (TCA) cycle to sustain mitochondrial function [16]. PI3K-AKT signaling is the most well-known mechanism for activating mTORC1 [17]. Besides, mTORC1 can be activated or inhibited by various signaling pathways directly or indirectly. For instance, 5′-AMP-activated protein kinase (AMPK) activated by energy shortage is a crucial inhibitor of mTORC1 [18]. What's more, transcription factor c-Myc and p53 also take part in metabolic reprogramming through transcriptional regulation of metabolism-related genes [19,20]. Based on multiple regulatory mechanisms, expression or activity of the enzymes involved in glucose, amino acids and fatty acids metabolism are altered, directly contributing to metabolic reprogramming [21]. What's more, the upregulation of various metabolic processes in cancer cells triggers accumulation of reactive oxygen species (ROS) [22]. Transcription factors nuclear factor erythroid 2related factor 2 (NRF2) and HIF-1 play key roles in maintenance of redox homeostasis, keeping ROS in an appropriate level to promote tumor growth rather than inducing damage [23,24].
Under nutrient rich conditions, activation of mTORC1 supports cancer cells growth. In periods of cellular stress, low levels of amino acids or absent ATP induces mTORC1 inhibition, which subsequently activates a compensatory mechanism named autophagy [25]. Autophagy is a highly regulated pathway essential for cell survival in nutrient-deprived conditions, complementing the classical pathways like glycolysis. Autophagy supplies amino acids by inducing degradation of macromolecules and organelles in lysosome, thereby providing intracellular amino acids supply to fuel the TCA cycle, gluconeogenesis and protein synthesis [26]. However, the interplay between autophagy and glycolysis seems to be complex. Activation of autophagy has been observed to enhance glycolysis [27]. Deficiency of mitophagy can induce mitochondrial dysfunctions, enhancing glycolysis and Warburg effect [28]. Additionally, studies have found that oxidative stress induced by cancer cells can promote aerobic glycolysis and autophagy in cancer associated fibroblasts to obtain recycled nutrients from cancer associated fibroblasts. This phenomenon is called "Reverse Warburg Effect" [29]. Therefore, both the anabolic pathways, such as glycolysis, and the catabolic pathways, such as autophagy, interplay with each other, together contribute to cancer metabolism and supporting cellular growth. Taken together, abnormal alterations of multiple signaling pathways, transcription factors and metabolic pathways synergistically lead to metabolic reprogramming in cancer cells.

Ubiquitination and deubiquitination
Ubiquitination is an ATP-dependent cascade process ligating ubiquitin, a ubiquitously expressed protein consisting of 76 amino acids, to a substrate protein [30]. Ubiquitin-activating enzymes (E1s) initially bind to ubiquitin for activation, and then transfer activated ubiquitin to ubiquitin-conjugating enzymes (E2s). Ubiquitin ligases (E3s) finally transfer ubiquitin from E2 to substrates [30]. According to the number of ubiquitin attaching to one lysine residue in protein, ubiquitination is divided into monoubiquitination (single ubiquitin) and polyubiquitination (a chain of ubiquitin) [31]. In the polyubiquitination chain, ubiquitin can be attached via 7 lysine residues (K6, K11, K27, K29, K33, K48, and K63) or the first methionine (M1) [32]. Different types of ubiquitination lead to disparate fates of substrate proteins. K48-linked polyubiquitination is the most widely studied type, which mainly labels proteins for 26S proteasomemediated recognition and degradation [32]. K48-linked polyubiquitination also has proteasome independent functions, including regulation of signaling events and transcription, which are possibly determined by the length of the ubiquitin chain [33][34][35]. K11-linked polyubiquitination is also associated with proteolysis [32]. Ubiquitin-proteasome system is involved in the degradation of more than 80% of proteins in cells [36]. K63-linked polyubiquitination is involved in signaling assemblies [32]. E3 ligases play a key role in the whole process of ubiquitination because of their specificity for substrates. In human genome, there are approximately 1000 E3 ligases, which can be divided into the homology to E6AP C terminus (HECT) domain-containing E3s, the RING-between-RING (RBR) family E3s and the really interesting new gene (RING) finger domain-containing E3s [37]. Deubiquitination is catalyzed by deubiquitinating enzymes (DUBs) to remove ubiquitin from ubiquitinated proteins, thus reversing the ubiquitination process [7]. About 100 DUBs fall into seven subgroups: the ubiquitin-specific proteases (USPs), the ubiquitin C-terminal hydrolases (UCHs), the ovarian tumor proteases (OTUs), the Machado-Josephin domain proteases (MJDs), the JAB1/MPN+/ MOV34 (JAMM) domain proteases, the monocyte chemotactic protein-induced proteins (MCPIPs), and the motif interacting with ubiquitin-containing DUB family (MINDY) [10]. Dynamic conversion between ubiquitination and deubiquitination is closely related to various cellular functions and thus, its dysregulation results in multiple disease, such as neurodegenerative diseases and cancer [38]. Understanding of ubiquitination and deubiquitination may provide novel insights into the treatment of these diseases.

Ubiquitination of mTOR
Aberrant activation of mTORC1 is considered as a key feature of metabolic reprogramming. mTORC1 is a complex consisting of mTOR, Raptor, mLST8, PRAS40 and DEPTOR [39]. mTOR is an evolutionarily conserved serine/threonine protein kinase in the PI3K-related kinase superfamily, responsible for the catalytic activity of mTORC1 [40]. Translocation of mTORC1 to lysosome is the premise for its subsequent activation, identified as a critical step in the activation of mTORC1 signaling [41]. Activated RagA is thought to be the main participator in the re-localization of mTORC1 to the lysosomes in amino acid-stimulated cells [41]. Studies have found that E3 ligase TRAF6, which is upregulated in cancer cells, can mediate K63-linked polyubiquitination of mTOR by interacting with p62 under the stimulation of amino acids, promoting the translocation of mTORC1 to the lysosomes and subsequent activation ( Fig. 1) [42]. In addition, decreased K48-linked ubiquitination of mTOR by E3 ligase FBX8 and FBXW7 alleviates proteasome-dependent degradation of mTOR, exerting an oncogenic effect in cancer as well [43,44]. Reduced mTOR ubiquitination is also linked to therapy resistance in cancer. Everolimus is a mTOR inhibitor used in breast cancer patients. Following downregulated phosphorylation of mTOR induced by depletion of dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2), ubiquitination and degradation of mTOR diminish, resulting in everolimus resistance [45]. Nonthermal plasma exerts anti-tumor effect by inducing RNF126 mediated K48-linked polyubiquitination and degradation of mTOR [46]. However, when faced with mitochondrial stress, E3 ligase PARKIN targets mTOR for ubiquitination, which maintains mTORC1 activity instead of affecting mTOR stability, thereby enhancing cell survival [47]. DUB USP9X can negatively modulate mTOR function and mTORC1 activity without changing mTOR protein level [48]. Therefore, different types of ubiquitination and deubiquitination play diverse roles in the regulation of mTOR function.

Ubiquitination of raptor
Raptor is the regulatory protein of mTORC1, maintaining the correct subcellular localization of mTORC1 and allowing the binding of mTORC1 with substrates [49]. DDB1-CUL4 E3 ligase complex is essential for maintaining mTORC1 stability by ubiquitinating Raptor. Displacement of DDB1-CUL4 complex by DUB UCH-L1 can remove K63-linked poly-ubiquitin chains on Raptor, bringing about reduced mTORC1 [49].

Ubiquitination of mLST8
mLST8, also called GβL, is a component of both mTORC1 and mTORC2. mLST8 is associated with the catalytic domain of mTOR and stabilizes its kinase activation loop [50]. mLST8 can be ubiquitinated by TRAF2 through K63-mediated linkage, which breaks the interaction between mLST8 and SIN1 in mTORC2, giving rise to elevated formation of mTORC1. This process can be reversed by DUB OTUD7B, leading to increased mTORC2 formation [50]. The study highlighted the role of ubiquitination and deubiquitination in the balance and competence between mTORC1 and mTORC2 signaling under various conditions.

Ubiquitination of RagA
As we mentioned above, activated RagA plays a key role in the re-localization of mTORC1 to the lysosomes and subsequent activation of mTORC1 [41]. Study found that downregulation of lysosomal E3 ligase RNF152 protected cells from autophagy [58]. RNF152 modifies RagA by K63-linked polyubiquitination and promotes recruitment of RagA inhibitor GATOR1, thus inducing RagA inactivation. Then mTORC1 is released from the lysosomal surface, giving rise to blockade of mTORC1 signaling pathway [58]. Moreover, K63-linked polyubiquitination of RagA can also be mediated by E3 ligase SKP2, which exerts a similar effect with RNF152 [59].

Ubiquitination of GATORs
GATOR1 is a complex consisting of DEPDC5, NPRL2 and NPRL3, while GATOR2 consists of Mios, WDR24, WDR59, Seh1L and sec13. GATOR2 negatively regulates DEPDC5 in GATOR1, which acts as an inhibitor of RagA. Thus, GATOR2 exerts a promoting effect on RagA. Oncogenic E3 ligase CUL3-KLHL22 was observed to mediate K48-linked polyubiquitination of DEPDC5 and target DEPDC5 for degradation under the stimulation of amino acids and promote mTORC1 activation in tumor [60].

Ubiquitination of Rheb
GTP-bound Rheb is the activator of mTORC1 after translocation of mTORC1 to the lysosome. Monoubiquitination of Rheb by the lysosomal E3 ligase RNF152 can enhance its interaction with the tuberous sclerosis complex (TSC) complex, the major inhibitor of Rheb, and can decrease mTORC1 activation [61]. Following phosphorylation by AKT, USP4 can deubiquitinate Rheb to reverse the action of RNF152. Therefore, the dynamic change of ubiquitinating state of Rheb is associated with mTORC1 activation and tumor growth [61].

Ubiquitination of PI3K
PI3K-mTORC1 signaling is the crucial signaling pathway that governs metabolic reprogramming and tumor cell growth. PI3K can be activated under the stimulation of growth factors. Activated PI3K phosphorylates PIP2, converting PIP2 to PIP3, which recruits 3-phosphoinositide-dependent protein kinase 1 (PDK1) and Akt to the membrane. PDK1 subsequently activates AKT, a negative regulator of TSC complex, and subsequently activating mTORC1 [69]. Activation of PI3K-mTORC1 signaling can exert various effects on metabolic process and play a key role in the regulation of tumor metabolism [69]. PI3K is a dimeric enzyme composed of a catalytic subunit (p110α, p110β, or p110γ) and a regulatory subunit (p85α, p85β, p55α, p55γ, or p50α) [70]. A dynamic cycle of proteasome-dependent degradation and resynthesis of PI3Kp110α were observed in activation of PI3K signaling. NEDD4L E3 ligase catalyzes free PI3Kp110α for ubiquitination, leading to its proteasome-dependent degradation and maintenance of PI3K signaling [70]. TRAF6 E3 ligase promotes activation of PI3K pathway in cancer by nonproteolytic polyubiquitination of PI3K catalytic subunit p110α [70]. TRAF6 also directs PI3K recruitment to TGF-β receptor via K63-linked polyubiquitination of PI3Kp85α, which is essential for TGF-β-induced activation of PI3K signaling [71]. What's more, PI3K regulatory subunit p85α can be ubiquitinated by E3 ligase MKRN2 and E3 ligase complex HSP70-CHIP through K48-mediated linkage, bringing about proteolysis of PI3Kp85α and downregulation of PI3K signaling in cancer [72,73]. Dephosphorylated free p85β is ubiquitinated by FBXL2 for proteolysis. Decreased free p85β reduces its competition with p85-p110 heterodimers for docking sites on cell membrane, thus upregulating PI3K signaling [74]. Therefore, both the catalytic subunit and the regulatory subunit can be ubiquitinated, exerting various effects on PI3K signaling.

Ubiquitination of PDK1
PDK1 phosphorylates and activates AKT, transducing signal from activated PI3K to AKT. Attenuated ubiquitination and degradation of PDK1 are related to chemoresistance in ovarian cancer [75]. Monoubiquitination of PDK1 in cancer cell lines can be reversed by USP4 catalyzation, the function of which still remains unclear [76].

Ubiquitination of AMPK
AMPK is an inhibitor of mTORC1 by phosphorylating Raptor and activating TSC2. As an energy sensor, AMPK is crucial in maintenance of NADPH and ATP level in response to reduced intracellular ATP. AMPK is composed of catalytic α and regulatory β and γ subunits [118]. E3 ligase complex MAGE-A3/6-TRIM28 and E3 ligase CRL4A catalyze ubiquitination of AMPKα and target it for degradation, thus reducing autophagy and altering cancer metabolism [118,119]. UBE2O, an atypical ubiquitin enzyme with both E2 and E3 activities, ubiquitinates AMPKα2 for degradation [120]. CRL4 catalyzed ubiquitination also directs AMPKγ proteolysis [121]. GID ubiquitin ligase mediates ubiquitination and degradation of AMPK as well, leading to decreased autophagy and increased mTOR activity [122]. Ubiquitination of AMPKα can be reversed by USP10 to remove the ubiquitin chain from AMPKα and promote AMPK activation [122].

Ubiquitination of KRAS
As a common mutated oncogene driving tumorigenicity in pancreatic, colon and lung cancers, KRAS enhances expression of glucose transporter type 1 (GLUT1) and controls glycolysis and glutamine metabolism in cancer cells, which is considered to be associated with metabolic reprogramming in primary invasive cancers [123]. Monoubiquitination and diubiquitination of KRAS elevate its GTP loading ability [124]. CUL3-based E3 ligase complex can mediate polyubiquitination and degradation of KRAS [125]. KRAS4B, an alternative splicing of KRAS gene, is targeted by E3 ligase NEDD4 for ubiquitination and proteolysis. However, activated KRAS signaling upregulates NEDD4 expression and prevents NEDD4-mediated KRAS ubiquitination. This in return promotes NEDD4 catalyzed degradation of PTEN to trigger tumor growth [126]. Therefore, modification of signaling molecules by ubiquitination can exert various effects in signaling pathways, thereby regulating metabolic reprogramming in cancer cells.

Ubiquitination of SREBP1
SREBP1 is a transcription factor associated with lipogenic genes [219]. mTORC1 signaling induces enhanced activity of SREBP1 to meet the requirements for fatty acid in proliferating cancer cells [220]. Activation of SREBP1 upregulates fatty acid synthesis as well as lipid import from extracellular space [220]. After phosphorylated by GSK-3, SREBP1 is prone to ubiquitination by E3 ligase FBXW7 and degradation via the ubiquitin-proteasome system [221,222]. Deacetylation of SREBP by SIRT1 also enhances its ubiquitination and proteolysis [223]. RNF139 can ubiquitinate precursor forms of SREBP1, thereby preventing SREBP1 synthesis [224].

Ubiquitination of ULK1
In nutrient-deprived conditions, inhibition of mTORC1 subsequently activates autophagy, a highly regulated pathway essential for cell survival [25]. Autophagy complements amino acids by inducing degradation of macromolecules and organelles in lysosomes [26]. ULK1 complex, composed of the ULK1, ATG13 and FIP200, is directly regulated by mTORC1 and is required for autophagy induction. TRAF6 catalyzed K63-linked polyubiquitination of ULK1 can stabilize ULK1 to promote activation of autophagy, which is related to drug resistance in chronic myeloid leukemia patients [225]. Activating molecule in BECN1-regulated autophagy protein 1 (AMBRA1) is a cofactor that interacts with Beclin-1 to regulate autophagy. Decreased phosphorylation of AMBRA1 caused by mTORC1 inactivation will promote its interaction with TRAF6 to upregulate polyubiquitination of ULK1, thereby potentiating autophagy initiation [226]. TRIM16 and TRIM32 also target ULK1 for K63-linked polyubiquitination, which stabilizes ULK1 and increases its phosphorylating activity, respectively [227,228]. DUB USP1 hydrolyzes the K63-linked ubiquitin chain on ULK1 [229]. Downregulation of USP24 also enhances ULK1 ubiquitination, thereby increasing protein stability and kinase activity of ULK1 [230]. These studies indicate the shift between ubiquitinated ULK1 and deubiquitinated ULK1 is essential for autophagy initiation.
Ubiquitination was also observed to be essential in the regulation of autophagy threshold during autophagy progression. NEDD4L-mediated ULK1 ubiquitination via K27 and K29-linkage assembly induces its proteolysis [231]. Decreased level of ULK1 activates transcription of ULK1 to maintain its basal protein level. Newly synthesized ULK1 will be deactivated by mTOR to ensure a safe threshold of autophagy [231]. CUL3-KLHL20 complex can also downregulate autophagy by targeting activated ULK1 for ubiquitination and proteolysis [232]. ULK1 can be deubiquitinated by USP20, which prevents it from degradation, maintaining its basal level required for the initiation of autophagy [233]. In prolonged starvation, the interaction between USP20 and ULK1 reduces to terminate autophagy [233]. In conclusion, ubiquitination and deubiquitination play essential functions in both autophagy initiation and autophagy progression. Modulating ubiquitination might be an effective treatment for chemoresistant patients with enhanced autophagy in cancer cells.
AMBRA1 is degraded via the action of E3 ligase CUL4 under normal conditions. Activation of ULK1 disassociates CUL4 from AMBRA1, causing stabilization of AMBRA1 to promote autophagy. Disassociation of AMBRA1 with CUL4 can promote AMBRA1 binding to CUL5 to inhibit CUL5-mediated DEPTOR degradation, thereby inducing autophagy [55]. CUL4 can re-associate with AMBRA1 to promote its proteolysis when autophagy terminates, thus regulating autophagy response [55]. What's more, E3 ligase RNF2 can also catalyze K48linked ubiquitination and proteolysis of AMBRA1, thus downregulating autophagy [246]. Therefore, all the components of the Class III PI3K complex can be regulated by ubiquitination, which exerts important effects on autophagy.

Ubiquitination of WIPI2
WIPI2 is involved in an early step of the formation of preautophagosomal structures. mTORC1 can mediate phosphorylation of WIPI2. E3 ligase HUWE1 interacts with phosphorylated WIPI2 and catalyzes ubiquitination of phosphorylated WIPI2, which is subsequently targeted for proteolysis, thus inhibiting autophagy flux [247].

Ubiquitination of ATG4
ATG4 contributes to LC3 processing, playing an essential role in the phagophore expansion and autophagosome completion. E3 ligase RNF5 targets ubiquitination and degradation of ATG4B, thus limiting autophagy flux. When cell starves, RNF5 de-associates with ATG4B to induce autophagy [248].

Ubiquitination and glucose metabolism
Increased glucose uptake and enhanced glycolytic flux are metabolic characteristics of cancer cells, supplying subsidiary pathways to provide precursors for macromolecule synthesis. Activated AKT was observed to inhibit ubiquitination of HK1, the first rate-limiting enzyme in the glucose metabolism pathway, promoting glycolysis and glioblastoma progression [249]. HUWE1 mediated K63-linked ubiquitination of HK2 promotes its re-localization and activation, enhancing glycolysis and tumor growth (Fig. 2) [250]. TRAF6 mediated K63linked ubiquitination of HK2 directs HK2 degradation by autophagy, thereby negatively regulating glycolysis [251]. Deubiquitination of HK2 catalyzed by CSN5 can rescue it from degradation and enhance glycolytic flux during hepatocellular cancer metastasis [252]. Mitochondrial HK can also be ubiquitinated by PARKIN, inducing its proteasomal degradation [253].
Phosphofructokinase (PFK), the second rate-limiting enzyme in glycolysis, is a key regulator of glycolytic flux in cancer cells. Decreased A20 mediated ubiquitination and degradation of PFK liver type (PFKL) are related to increased glycolysis during hepatocellular carcinoma progression [254]. Phosphorylation of PFK1 platelet isoform (PFKP) by AKT can prevent it from TRIM21mediated ubiquitination and degradation, promoting aerobic glycolysis in glioblastoma cells [255].
Pyruvate kinase M2 (PKM2) is the third rate-limiting enzyme of glycolysis. Decreased CHIP catalyzed ubiquitination and degradation of PKM2 are associated with Warburg effect in ovarian cancer cells [256]. Downregulated ubiquitination of PKM2 by TRIM58 is related to progression of osteosarcoma [257]. E3 ligase PARKIN modifies PKM2 by ubiquitination to decrease its enzymatic activity without affecting its stability [258]. PKM2 deubiquitinated by USP7 can strengthen the protein stability of PKM2 [259]. USP20 can also hydrolyze the ubiquitin chain on PKM2, but the detailed function of this deubiquitination is unclear [260].
Ubiquitination of enzymes involved in the TCA cycle is also associated with cancer progression. Decreased UBR5-mediated ubiquitination of citrate synthase leads to citrate accumulation in hypoxia breast cancer cells, promoting cell migration, invasion, and metastasis [269]. HIF-1 activation under hypoxia condition can promote α-ketoglutarate dehydrogenase (α-KGDH) complex ubiquitination and proteolysis by SIAH2. Decreased α-KGDH activity inhibits glutamine oxidation and promotes glutamine-dependent lipid synthesis for tumor growth [270]. USP13 promotes ovarian cancer progression by deubiquitinating and upregulating α-KGDH [271].
Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the oxidative pentose phosphate pathway, an essential process producing ribose-5-phosphate and NAPDH from G6P. G6PD was observed to be ubiquitinated and degraded by VHL E3 ubiquitin ligase in podocytes [272]. VHL is tumor-suppressor protein [273]. But whether this regulation exists in cancer cells is unclear. Fructose-1,6-biphosphatase (FBP1) is a rate-limiting enzyme of gluconeogenesis. MAGE-TRIM28 mediated ubiquitination and degradation of FBP1 in hepatocellular carcinoma promotes Warburg effect and cancer progression [274]. Phosphoenolpyruvate carboxykinase 1 (PEPCK1) is another rate-limiting enzyme in gluconeogenesis. High glucose in diabetes stimulates PEPCK1 acetylation, which promotes UBR5 mediated ubiquitination and degradation of PEPCK1 [275]. Studies have found that GLUT1 can be ubiquitinated for degradation in diabetes [276]. E3 ubiquitin ligase Malin mediated ubiquitination of glycogen debranching enzyme (AGL) is associated with Lafora and Cori's disease [277]. But whether ubiquitination of GLUT1, PEPCK1 and AGL participates in tumor progression is still unknown. What's more, ubiquitination and deubiquitination of other cancer associated metabolic enzymes is still unknown, such as enolase in glycolysis, isocitrate dehydrogenase in the TCA cycle, glycogen phosphorylase in glycogen metabolic process, and pyruvate carboxylase in gluconeogenesis. The relationship between their specific E3 ligases and DUBs and oncogenesis still need exploration.

Ubiquitination and fatty acid metabolism
Fatty acids synthesis is necessary for membrane biosynthesis in proliferating tumor cells. CUL3-KLHL25 E3 ligase can inhibit lipid synthesis and tumor growth by targeting ATP citrate lyase (ACLY) for ubiquitination and proteolysis [278]. However, USP13 and USP30 can mediate deubiquitination of ACLY, increasing the stability of ACLY to promote development of ovarian cancer and hepatocellular carcinoma, respectively [271,279]. TRB3-COP1 mediates proteolysis of acetyl-coenzyme A carboxylase (ACC) in ubiquitin-dependent manner, inhibiting fatty acid synthesis and stimulating lipolysis [280]. But in breast cancer cells, ACC-alpha (ACCA) interacts with AKR1B10, which prevents ACCA from ubiquitination and proteolysis, thereby promoting de novo fatty acid synthesis and enhancing tumor growth [281]. E3 ligase COP1 can ubiquitinate fatty acid synthase (FASN) with SHP2 as an adapter [282]. E3 ligase SPOP mutation, which is common in prostate cancer, inhibits its ubiquitination of FASN. Increased FASN triggers lipid accumulation and promotes prostate cancer progression [283]. AKT activation promotes deubiquitination of FASN by the USP2a and increased lipogenesis, which promotes hepatocarcinogenesis [284,285].
Squalene epoxidase (SQLE), which catalyzes the first oxygenation reaction of cholesterol biosynthesis, can be ubiquitinated and degraded by E3 ligase MARCH6 under stimulation of sterol [291]. Additionally, E3 ligase MYLIP can modulate cellular cholesterol uptake by ubiquitinating LDL receptor which is responsible for cholesterol import [292]. But whether ubiquitination of SQLE and LDL receptor participate in cancer progression is unknown. In addition, ubiquitination and deubiquitination of other enzymes participating in fatty acid metabolism, such as Carnitine O-palmitoyltransferase 1 (CPT1), and their relationship with cancer progression have not been studied yet.

Ubiquitination and amino acid metabolism
In cancer cells, glutamine serves as another important carbon source for the TCA cycle to sustain mitochondrial ATP production [1]. Glutamine uptake increases dramatically in cancer cells. NEDD4L-depleted cancer cells have enhanced neutral amino acid transporter B (ASCT2) stability and glutamine uptake to fuel the mitochondrial metabolism [293]. Promotion of RNF5targeted ubiquitination and degradation of glutamine carrier proteins ASCT2 and SLC38A2 can improve responsiveness of breast cancer cells to Paclitaxel treatment [294]. Glutamine can be converted via GLS to glutamate, which can subsequently be converted to αketoglutarate to fuel the TCA cycle. Succinylation of GLS suppresses its K48-linked ubiquitination and degradation, stabilizing GLS and promoting glutaminolysis in cancer cells [295]. Study also found the function of supranutritional dose of selenite in suppressing tumor progression by promoting APC/C-CDH1 mediated GLS1 ubiquitination and degradation [296]. Ubiquitination of other enzymes involved in glutamine metabolism, such as glutamate dehydrogenase 1 (GLUD1), hasn't been studied.
3-phosphoglycerate, an intermediate product of glycolysis, can be converted to serine by D-3phosphoglycerate dehydrogenase (PHGDH). This conversion is subsequently associated with formate production for nucleotide synthesis. Downregulated PARKIN in cancer suppresses ubiquitination of PHGDH and enhances its stability and protein level, thereby activating serine synthesis and promoting cancer progression [297]. Serine hydroxymethyltransferase 1 (SHMT1) is involved in the conversion of serine to glycine. K48-linked ubiquitination of SHMT1 mediates its degradation in the cytoplasm. K63-linked ubiquitination of SHMT1 by UBC13 in the nucleus promotes its nuclear export and prevents it from degradation, promoting tumor progression [298]. What's more, dysregulation of aspartate and arginine metabolism is also associated with cancer progression [299]. However, ubiquitination and deubiquitination of the enzymes participating in aspartate and arginine metabolism haven't been studied and are worth attention in the future.

Conclusions
In the past decades, extensive efforts have been made to clarify the molecular mechanisms associated with metabolic reprogramming in cancer. In this review, we highlight the roles of ubiquitination and deubiquitination as modulators of cancer metabolism. Facing metabolic stresses, such as hypoxia, ubiquitination and deubiquitination in cancer cells can be abnormally regulated [10,270]. On the other hand, dysregulated ubiquitination and deubiquitination play nonnegligible roles in cancer metabolism by involving in the regulation of metabolic reprogramming related signaling pathways, transcription factors as well as metabolic enzymes. For instance, hypoxia induces E3 ubiquitin ligase SIAH2 mediated ubiquitination and proteolysis of α-KGDH, inhibiting glutamine oxidation and promoting glutaminedependent lipid synthesis to promote tumor growth [270]. Therefore, the interactions between ubiquitination/deubiquitination and cancer metabolism are complex and require more studies. Most studies have focused on the involvement of ubiquitination and deubiquitination in the regulation of signaling pathways and transcription factors, while ubiquitination and deubiquitination of the enzymes involved in glucose, fatty acid and amino acid metabolism are worth more attention in the future.
In the regulation of cancer metabolism and tumor progression, the E3 ubiquitin ligases/DUBs-substrates network is of high complexity. Single E3 ubiquitin ligase or DUB can target numerous substrates, and one molecule can be regulated by multiple E3 ubiquitin ligases or DUBs. For example, FBXW7 acts as a tumor suppressor by targeting mTOR, HIF-1α, c-Myc and SREBP1 for degradation [43,136,160,222]. However, when facing DNA damage, elevated FBXW7 mediates proteasomal degradation of p53, leading to radiotherapy resistance [300]. Amino acids can stimulate subcellular localization of TRAF6 to lysosomes for subsequent K63-linked polyubiquitination and activation of mTOR signaling [42]. However, in starvation induced autophagy, TRAF6 mediates K63-linked polyubiquitination of ULK1, which leads to stabilization of ULK1 and activation of autophagy [225]. Therefore, E3 ligases and DUBs act in a context-dependent manner. Their exact roles in cancer may vary according to their substrates, tissues types, tumor stages, or different metabolic conditions. The study of ubiquitination and deubiquitination in cancer still has a long way to go. For example, whether metabolite levels within cancer cells act as modulators of ubiquitination is ambiguous. Importantly, development of specific drugs that disrupt or enhance specific E3 ligases/DUBs-substrates interactions holds promise for more efficient and less toxic therapeutics.
What's more, we have observed that decreased ubiquitination and increased stability of the metabolic related molecules, such as PDK1, NRF2, ULK1 and phosphoglycerate kinase 1, are associated with chemoresistance in various cancers. Thereby, modulating the activity of E3 ligases or DUBs could be exploited as a potential strategy for controlling chemoresistance in cancer treatment. Furthermore, various E3 ligases and DUBs have been already identified as potential targets for cancer therapy. Actually, many E3 ligases serve as tumor suppressors by catalyzing ubiquitination and degradation of metabolic related proteins which play oncogenic roles in cancers, indicating that drugs enhancing activities or expression of these E3 ligases should also be emphasized in further researches.
In conclusion, ubiquitination and deubiquitination are suggested to be essential regulators of metabolic reprogramming in cancer cells, demanding more studies in the future with the aim of improving cancer therapy.