ALDH2 Suppresses Glycolysis in Hepatocellular Carcinoma via TRIM21-Mediated GLUT1 Degradation

ALDH2 Suppresses Glycolysis in Hepatocellular Carcinoma via TRIM21-Mediated GLUT1 Degradation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article ALDH2 Suppresses Glycolysis in Hepatocellular Carcinoma via TRIM21-Mediated GLUT1 Degradation Huiyong Yin, Shanshan Zhong, Lili Zhang, Yongqiang Wang, Ningning Liang, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7102103/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Aldehyde dehydrogenase 2 (ALDH2) detoxifies alcohol-derived acetaldehyde and lipid aldehydes from lipid peroxidation. A single nucleotide polymorphism of ALDH2 rs671, representing 30-50% East Asians and featuring ALDH2 deficiency, is associated with increased risk of hepatocellular carcinoma (HCC), but the underlying mechanism remains unclear. Here we demonstrate a non-catalytic role of ALDH2 in regulating glucose metabolism through post-translational control of GLUT1 stability. Using ALDH2 knockout and rs671 knock-in mice, we show that ALDH2 interacts with GLUT1 and promotes its K48-linked ubiquitination at lysine 256 via recruitment of E3 ligase TRIM21. ALDH2 deficiency reduces GLUT1 ubiquitination, stabilizes GLUT1 protein, enhances glycolytic flux, and promotes HCC development. These effects occur independently of alcohol exposure. Pharmacological inhibition or genetic silencing of GLUT1 reverses the tumor-promoting effect of ALDH2 deficiency in both xenograft and DEN-induced models. These findings reveal an unrecognized metabolic function of ALDH2 and nominate GLUT1 as a tractable vulnerability in ALDH2-deficient HCC. Biological sciences/Cancer/Gastrointestinal cancer/Liver cancer/Hepatocellular carcinoma Biological sciences/Cell biology/Post-translational modifications/Ubiquitylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, ranks among the leading causes of cancer-related death worldwide 1 , 2 . Despite advances in diagnosis and treatment, its prognosis remains poor, mainly due to late-stage detection and limited therapeutic options 1 . A deeper understanding of the molecular mechanisms driving HCC is critical for developing more effective targeted therapies. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that catalyzes the oxidation of acetaldehyde, a byproduct of alcohol metabolism, and detoxifies lipid peroxidation–derived aldehydes 3 , 4 . A common single nucleotide polymorphism (SNP), rs671, results in a glutamic acid–to–lysine substitution at position 487 and is present in approximately 30–50% of East Asians and 8% of the global population 5 . This variant markedly reduces ALDH2 enzymatic activity and protein stability, leading to reduced ALDH2 expression 6 – 8 . Clinically, ALDH2 expression is significantly downregulated in HCC and correlates with poor prognosis, particularly in individuals with alcohol exposure 8 – 11 . Paradoxically, rs671 carriers tend to consume less alcohol due to adverse reactions, such as facial flushing 12 , 13 , suggesting that ALDH2 may also have alcohol-independent role in liver tumorigenesis. Our previous work has demonstrated non-catalytic functions of ALDH2 in lipid metabolism 14 , 15 . however, whether and how ALDH2 contributes to HCC progression independently of alcohol metabolism remains unknown. Metabolic reprogramming is a hallmark of cancer, and many tumors rely on elevated aerobic glycolysis (the Warburg effect) to support rapid proliferation 16 17 . Increased glucose uptake is a prerequisite for this phenotype and is often driven by elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) 18 . GLUT1 is frequently upregulated in HCC and is associated with poor clinical outcomes 19 , 20 , highlighting its potential as a therapeutic target. However, the upstream mechanisms that regulate GLUT1 stability in HCC remain poorly understood. Here, we identify a previously unrecognized, alcohol-independent role of ALDH2 in suppressing HCC progression through post-translational regulation of GLUT1. Mechanistically, ALDH2 promotes the recruitment of the E3 ubiquitin ligase TRIM21 to GLUT1, facilitating K48-linked polyubiquitination at lysine 256 (K256) and subsequent proteasomal degradation. Loss of ALDH2—via genetic deletion or the rs671 variant—disrupts this regulatory axis, resulting in GLUT1 accumulation, enhanced glycolytic flux, and accelerated tumor growth. Notably, pharmacological inhibition of GLUT1 with BAY-876 significantly suppresses tumor development in both xenograft and DEN-induced HCC mouse models with ALDH2 deficiency, highlighting a potential therapeutic strategy targeting metabolic vulnerabilities in ALDH2-deficient liver cancer. Results ALDH2 is downregulated in hepatocellular carcinoma and predicts poor prognosis To investigate the clinical relevance of ALDH2 in HCC, we analyzed its transcript levels in publicly available datasets. Both the TCGA-LIHC and GSE14520 cohorts revealed a significant reduction in ALDH2 mRNA expression in HCC tumor tissues compared to adjacent normal liver tissues (Fig. 1 A–B). Kaplan–Meier survival analysis further demonstrated that low ALDH2 expression was associated with reduced overall survival in HCC patients (Fig. 1 C). These findings were validated at the protein level by immunoblotting of 24 paired human HCC and adjacent non-tumor liver tissues, which confirmed a marked downregulation of ALDH2 in tumors (Fig. 1 D–E). To evaluate the functional role of ALDH2 in HCC, we utilized Aldh2 global knockout mice and subjected them to diethylnitrosamine (DEN)-induced liver tumorigenesis. Efficient ALDH2 knockout in liver tissues was confirmed by immunoblotting (Fig. 1 F). Compared to wild-type controls, AKO mice exhibited significantly increased tumor burden, as evidenced by elevated liver-to-body weight ratios, increased tumor number, and greater maximum tumor volume (Fig. 1 G–J and Supplemental Fig. 1F). To extend these findings to a clinically relevant genetic context, we utilized Aldh2 rs671 knock-in mice (E504K), which model the common East Asian loss-of-function variant 8 . Consistent with the knockout model, rs671 mice exhibited reduced hepatic ALDH2 protein expression (Supplementary Fig. 1A) and developed more numerous and larger liver tumors following DEN administration, with a shifted tumor number distribution (Supplementary Fig. 1B–E). Together, these results establish that ALDH2 deficiency promotes HCC development in an alcohol-independent manner. ALDH2 loss enhances glucose metabolism in HCC To investigate the metabolic consequences of ALDH2 deficiency in HCC under non-alcoholic conditions, we performed untargeted metabolomic profiling of liver tissues from WT and AKO mice following DEN treatment, using a high-resolution mass spectrometry-based platform previously established in our laboratory 21 . Unsupervised clustering of significantly altered metabolites revealed marked changes in carbohydrate and nucleotide metabolism in AKO livers compared to WT controls (Fig. 2 A). Pathway enrichment analysis further indicated upregulation of the Warburg effect and the tricarboxylic acid (TCA) cycle pathways (Fig. 2 B), suggesting a metabolic shift toward enhanced glucose utilization in ALDH2-deficient HCC livers. To further characterize these alterations, we conducted [U-¹³C₆]-glucose tracing in vivo (Fig. 2 C). Compared with WT controls, both AKO and rs671 knock-in livers showed significantly increased ¹³C enrichment in glycolytic intermediates—pyruvate, lactate, and alanine—indicating elevated glycolytic flux (Fig. 2 D–E). Additionally, enhanced labeling of TCA cycle–derived metabolites, such as citrate, proline, fumarate, malate, aspartate, and glutamate, was observed in both models (Supplementary Fig. 2A–B), indicating increased glucose oxidation through mitochondrial metabolism. These findings were further validated in vitro using HCC cells cultured with [U-¹³C₆]-glucose, ALDH2 knockdown led to elevated incorporation of ¹³C into both secreted glycolytic products and intracellular TCA intermediates, including citrate, malate, and glutamate (Supplementary Fig. 2C–E). To assess whether altered glucose uptake—a critical initiating step of glycolysis 22 – 25 —underlies this phenotype, we measured 2-NBDG-6-phosphate (2-NBDG) uptake. ALDH2 knockdown significantly enhanced glucose uptake in LPC-H12 cells (Fig. 2 F). Consistently, ALDH2-deficient cells consumed more glucose from the culture medium (Fig. 2 G), supporting a state of increased glucose demand driven by ALDH2 loss. ALDH2 loss promotes GLUT1-driven glucose metabolic activation in HCC The observed increase in glucose flux prompted us to investigate the underlying mechanisms driving enhanced glucose metabolism in ALDH2-deficient HCC. Given that glucose transporter 1 (GLUT1) is a key rate-limiting factor for glucose uptake in cancer cells, we examined its expression across multiple models. Immunoblotting revealed markedly elevated GLUT1 protein levels in DEN-induced tumors from ALDH2 knockout and rs671 knock-in mice compared to wild-type controls (Fig. 3 A and Supplementary Fig. 3A). Similarly, stable ALDH2 knockdown in LPC-H12 and Hepa1-6 HCC cells increased GLUT1 expression (Fig. 3 B). These findings were further supported by human clinical samples, in which tumors with reduced ALDH2 expression showed frequent upregulation of GLUT1 protein compared to matched adjacent non-tumor tissues (Fig. 3 C). Despite consistent GLUT1 protein upregulation, Glut1 mRNA levels remained unchanged in ALDH2-deficient mouse livers, rs671 mice, and HCC cell lines (Supplementary Fig. 3B–C), suggesting post-transcriptional regulation. To further examine the impact of the rs671 variant on GLUT1 regulation in human HCC, we stratified 50 paired tumor samples by ALDH2 genotype (WT vs rs671) and performed immunoblotting. Quantification revealed higher GLUT1 protein levels in rs671 tumors compared to WT counterparts (Fig. 3 D and Supplementary Fig. 3D). Analysis of TCGA data corroborated our findings, showing that SLC2A1 (GLUT1) is significantly upregulated in HCC tumors relative to normal liver (Supplementary Fig. 3E). Across the patient cohort, ALDH2 and GLUT1 protein levels were inversely correlated (Fig. 3 E), supporting a conserved regulatory relationship in human HCC. As GLUT1-mediated glucose transport requires translocation to the plasma membrane, we next examined whether ALDH2 deficiency alters GLUT1 subcellular distribution. Fractionation experiments revealed enrichment of GLUT1 in the plasma membrane (PM) fraction of ALDH2-deficient samples (Fig. 3 . F-G and Supplemental Fig. 3G), a finding further supported by immunofluorescence in ALDH2-knockdown HCC cells (Supplemental Fig. 3F). To determine whether GLUT1 is functionally required for the metabolic phenotype induced by ALDH2 loss, we treated cells with BAY-876, a selective GLUT1 inhibitor 26 . BAY-876 administration significantly reduced glucose consumption (Fig. 3 H), attenuated 13 C incorporation from [U-¹³C₆]-glucose into glycolytic and TCA intermediates (Fig. 3 I), and suppressed lactate secretion (Fig. 3 J), confirming the dependence of enhanced glucose metabolism on GLUT1 activity. In addition, patients with high GLUT1 expression exhibited significantly poorer overall survival (Supplementary Fig. 3H). To further evaluate the functional relevance of GLUT1 in hepatoma cells, we performed siRNA-mediated knockdown in parental LPC-H12 cells. GLUT1 silencing markedly suppressed cell proliferation, confirming its role as a driver of HCC cell growth independent of ALDH2 status (Supplementary Fig. 3I). Consistently, pharmacological inhibition of GLUT1 using the selective inhibitor BAY-876 resulted in a dose-dependent reduction in cell proliferation (Supplementary Fig. 3J), further supporting the therapeutic potential of targeting GLUT1. Moreover, patients with concurrent low ALDH2 and high GLUT1 expression had the poorest prognosis (Fig. 3 K), reinforcing the clinical relevance of the ALDH2–GLUT1 regulatory axis in HCC progression. ALDH2 deficiency stabilizes GLUT1 by reducing GLUT1 ubiquitination . To investigate how ALDH2 regulates GLUT1 protein abundance, we first assessed its effect on GLUT1 protein turnover. Cycloheximide (CHX) chase assays showed that GLUT1 degradation was significantly delayed in ALDH2-knockdown cells compared to controls, suggesting that ALDH2 promotes GLUT1 degradation (Fig. 4 A–B). To determine the relevant degradation pathway, we treated cells with either the proteasome inhibitor MG132 or the lysosome inhibitor chloroquine (CQ) (Supplementary Fig. 4A). MG132, but not CQ, effectively blocked GLUT1 degradation (Fig. 4 C–D; Supplementary Fig. 4B), suggesting that ALDH2 promotes GLUT1 turnover primarily through the ubiquitin–proteasome system. We next examined whether ALDH2 modulates GLUT1 ubiquitination. Immunoprecipitation of endogenous GLUT1 revealed reduced GLUT1 ubiquitination in both ALDH2-deficient liver tissues (Fig. 4 E–F) and ALDH2 knockdown LPC-H12 cells (Supplementary Fig. 4C), indicating that ALDH2 facilitates GLUT1 ubiquitination in both in vivo and in vitro. To confirm this in a defined system, we co-expressed V5-tagged GLUT1, HA-tagged ubiquitin, and either wild-type or rs671-mutant FLAG-tagged ALDH2 in 293T cells. Both ALDH2 constructs increased GLUT1 ubiquitination to a similar extent (Fig. 4 G), suggesting that ALDH2’s enzymatic activity is not required for this function. Given prior findings that that ALDH2 can bind to Rac2 and influence its degradation 27 , we hypothesized that ALDH2 may similarly interact with GLUT1 to facilitate its proteasomal degradation. Co-immunoprecipitation assays in 293T cells confirmed that both wild-type and rs671 ALDH2 can bind with GLUT1 (Supplementary Fig. 4D–E). This interaction was also detected in wild-type mouse liver tissues and LPC-H12 cells but was markedly reduced in ALDH2-deficient (AKO or rs671) samples, likely due to reduced ALDH2 levels (Fig. 4 H–I; Supplementary Fig. 4F). Importantly, endogenous ALDH2–GLUT1 binding was also observed in paired human HCC tumor and adjacent non-tumor tissues (Fig. 4 J). Together, these findings demonstrate that ALDH2 interacts with GLUT1 and promotes its ubiquitin-dependent proteasomal degradation. Loss of ALDH2—either via gene knockout or the rs671 variant—impairs GLUT1 ubiquitination, leading to its stabilization in liver and HCC cells. The ALDH2–TRIM21 complex catalyzes K48-linked ubiquitination of GLUT1 To identify the E3 ligase responsible for GLUT1 ubiquitination downstream of ALDH2, we performed mass spectrometry-based proteomic analysis following immunoprecipitation of ALDH2- and GLUT1-associated protein complexes. TRIM21 and MUL1 emerged as top candidate E3 ligases that potentially interact with both ALDH2 and GLUT1 (Fig. 5 A; Supplementary Fig. 5A–B). TRIM21 and MUL1 are conserved E3 ubiquitin-protein ligases known to regulate diverse signaling pathways including OCT1, p53, NF-κB, and HIF-1α 28 . To evaluate their role in GLUT1 regulation, we silenced each ligase in LPC-H12 cells with or without ALDH2 knockdown. GLUT1 levels increased upon the knockdown of either ligase, suggesting both contribute to GLUT1 degradation (Fig. 5 B). However, in co-transfected 293T cells, TRIM21 induced stronger GLUT1 ubiquitination than MUL1 (Fig. 5 C), a finding confirmed by endogenous ubiquitination assays (Supplementary Fig. 5C), supporting TRIM21 as the dominant E3 ligase in this context. Given that RING domain of TRIM21 is essential for its catalytic activity 29 , we used a RING-deficient TRIM21 mutant (ΔRING). Overexpression of wild-type TRIM21 increased GLUT1 ubiquitination, which was lost with the ΔRING mutant (Fig. 5 D), confirming that TRIM21 E3 ligase activity is required. To determine how ALDH2 facilitates this process, we assessed complex formation between ALDH2, TRIM21, and GLUT1. Co-immunoprecipitation in 293T cells showed that TRIM21 interacts with both ALDH2 and GLUT1 (Supplementary Fig. 5D–E), and that this interaction is preserved in the ALDH2 rs671 variant, indicating that enzymatic activity is dispensable. In liver tissues from wild-type, ALDH2-knockout, and rs671 mice, TRIM21-associated GLUT1 and ALDH2 levels were significantly reduced in ALDH2-deficient conditions (Fig. 5 E). Similar reductions were observed in ALDH2-knockdown LPC-H12 cells (Supplementary Fig. 5F). In human HCC specimens, TRIM21 co-immunoprecipitated both ALDH2 and GLUT1 in adjacent non-tumor tissues, while these interactions were diminished in matched tumor samples (Fig. 5 F–G). Reciprocal IP using anti-ALDH2 antibody further confirmed reduced interaction with TRIM21 and GLUT1 in tumors tissues (Supplementary Fig. 5G), supporting the idea that ALDH2 serves as a scaffold stabilizing the TRIM21–GLUT1 interaction. To directly test this mechanism, we performed in vitro ubiquitination assays using purified components. TRIM21 catalyzed polyubiquitination of GLUT1, which was abolished by ΔRING mutation (Fig. 5 H). Addition of recombinant ALDH2 further enhanced GLUT1 ubiquitination in a dose-dependent manner (Fig. 5 I), indicating that ALDH2 promotes TRIM21 activity. Next, we assessed linkage specificity. K48-linked polyubiquitin chains are canonical signals for proteasomal degradation 30 . TRIM21 selectively induced K48-linked—but not K63-linked—ubiquitination of GLUT1 (Fig. 5 J) and did not promote ubiquitination through other lysine linkages (K6, K11, K27, K29, K33; Supplementary Fig. 5H). Finally, both wild-type and rs671 ALDH2 enhanced TRIM21-mediated K48 ubiquitination to a similar degree (Fig. 5 K), demonstrating that this function is independent of ALDH2 enzymatic activity. Together, these results identify TRIM21 as the primary E3 ligase responsible for GLUT1 ubiquitination and reveal that ALDH2, through protein–protein interaction, facilitates TRIM21-mediated K48-linked ubiquitination of GLUT1, thereby promoting its degradation. Ubiquitination of GLUT1 at K256 is required for ALDH2-induced degradation To identify the specific ubiquitination site responsible for GLUT1 degradation, we performed mass spectrometry analysis and identified lysine 256 (K256) as a candidate ubiquitination site (Fig. 6 A). Sequence alignment showed that K256 is highly conserved across multiple species (Fig. 6 B), suggesting its potential functional importance. To evaluate its role in GLUT1 stability, we conducted cycloheximide (CHX) chase assays in cells expressing either wild-type or K256R-mutant GLUT1. The K256R mutant exhibited markedly increased stability compared to WT, indicating that K256 is a critical for GLUT1 degradation (Fig. 6 C). We next tested whether K256 is required for TRIM21-mediated ubiquitination. In 293T cells co-transfected with HA-tagged TRIM21 and V5-tagged GLUT1, the K256R mutation significantly impaired TRIM21-induced GLUT1 ubiquitination (Fig. 6 D). This was further validated using an in vitro reconstituted ubiquitination system, where recombinant TRIM21 and ALDH2 efficiently catalyzed polyubiquitination of WT-GLUT1, but not the K256R mutant (Fig. 6 E). Together, these findings identify K256 as the principal site for TRIM21-dependent ubiquitination of GLUT1. To assess the functional relevance of K256 ubiquitination in vivo, we generated subcutaneous xenograft models using Hep1-6 cells stably expressing either GLUT1^WT or GLUT1^K256R, with or without ALDH2 overexpression (Fig. 6 F). Immunoblotting confirmed successful GLUT1 expression overexpression in tumors (Fig. 6 G). ALDH2 overexpression significantly reduced tumor growth In GLUT1^WT tumors, but this effect was abolished in GLUT1^K256R tumors (Fig. 6 H–J), supporting a model in which ALDH2 suppresses tumorigenesis via K256-dependent ubiquitination and degradation of GLUT1. GLUT1 inhibition reverses tumor-promoting effects of ALDH2 deficiency To evaluate the therapeutic relevance of GLUT1 in ALDH2-deficient HCC, we first used a subcutaneous xenograft model with Hepa1-6 cells stably expressing shALDH2. Mice were treated with the selective GLUT1 inhibitor BAY-876 (4 mg/kg, oral gavage once daily), starting on day 7 post-injection (Fig. 7 A). ALDH2 knockdown significantly accelerated tumor growth, while BAY-876 treatment significantly suppressed tumor progression (Fig. 7 B–D), suggesting that ALDH2 deficiency drives tumorigenesis through a GLUT1-dependent mechanism amenable to pharmacological inhibition. To further confirm this dependency, we genetated a dual-knockdown model targeting both ALDH2 and GLUT1 in Hepa1-6 cells (Fig. 7 E–F). Genetic silencing of GLUT1 in the ALDH2-deficient cells significantly reduced tumor volume and weight (Fig. 7 G–I), consistent with the effect of BAY-876 treatment. These findings collectively support GLUT1 as a critical effector of ALDH2 loss in promoting tumor growth. To validate these results in a more physiologically relevant setting, we treated DEN-induced HCC mice (AKO and rs671) with BAY-876 beginning at week 28 (Supplementary Fig. 6A, F). No significant weight differences were observed post-treatment (Supplemental Fig. 7, A and D), indicating that the dosage was safe. BAY-876 significantly reduced tumor number and burden in both ALDH2 knockout and rs671 knock-in mice, while exerting minimal effects in wild-type controls (Supplementary Fig. 6B–D, G–I and Supplementary Fig. 7C, F). This selective response is likely due to lower baseline GLUT1 expression in wild-type livers, which limits the efficacy of GLUT1 inhibition in the presence of functional ALDH2. Stable isotope tracing further demonstrated that BAY-876 treatment attenuated the elevated glycolytic and TCA fluxes observed in ALDH2-deficient livers (Supplementary Fig. 6E, J; Supplementary Fig. 7B, E), confirming the mechanistic link between GLUT1 upregulation and metabolic reprogramming upon ALDH2 loss. Together, these data demonstrate that ALDH2 deficiency promotes tumorigenesis by enhancing GLUT1 expression and glucose metabolism, and that targeting GLUT1—genetically or pharmacologically—effectively suppresses tumor growth in ALDH2-deficient HCC. Discussion Although surgical and systemic therapies have improved survival in hepatocellular carcinoma (HCC), overall outcomes remain suboptimal compared to other common cancers 31 . A better understanding of tumorigenic mechanisms and the identification of precision biomarkers are critical to advance early diagnosis and precision therapies. Metabolic reprogramming, particularly the upregulation of glycolysis, is a hallmark of HCC that supports the energetic and biosynthetic demands. The ALDH2 rs671 variant is associated with poor HCC prognosis, especially in East Asian populations 4 , 8 , 32 . While most studies have focused on the accumulation of acetaldehydes due to reduced enzymatic activity of ALDH2 rs671, emerging evidence suggests that ALDH2 may also exert non-enzymatic functions independent of alcohol metabolism. In this study, we uncover previously unrecognized, non-catalytic function of ALDH2 in modulating glucose metabolism through regulation of GLUT1 stability. Mechanistically, ALDH2 facilitates the recruitment of the E3 ligase TRIM21, which catalyzes K48-linked polyubiquitination of GLUT1 at lysine 256, leading to its proteasomal degradation. ALDH2 deficiency—either by knockout or the rs671 variant—disrupts this interaction, resulting in GLUT1 stabilization and enhanced glycolytic activity in HCC cells (Fig. 8 ). These findings reveal a tumor-suppressive role of ALDH2 beyond its canonical mitochondrial function. Although metabolic reprogramming is well established in cancer biology, the upstream regulatory mechanisms governing glucose metabolism in HCC remain poorly defined 16 , 33 . Our prior work demonstrated that ALDH2 regulates lipid metabolism by stabilizing HMGCR, the rate-limiting enzyme in cholesterol biosynthesis 14 , 15 . While earlier studies linked ALDH2 to glucose metabolism in the context of alcohol exposure 9 , 34 – 37 , its role in non-alcoholic HCC has not been explored. Here, using metabolomics and [U-¹³C₆]-glucose tracing, we show that ALDH2 deficiency enhances glycolytic and TCA cycle fluxes via GLUT1 stabilization, supporting a Warburg-like metabolic phenotype. Our findings also show translational significance. GLUT1 is frequently overexpressed in HCC and correlates with poor clinical outcomes 20 , 38 – 41 . We demonstrate that ALDH2 deficiency increases GLUT1 protein levels and that genetic or pharmacological inhibition of GLUT1 suppresses tumor growth in ALDH2-deficient models. These results position ALDH2 and GLUT1 expression levels as potential prognostic markers and highlight GLUT1 as a therapeutic vulnerability, particularly in patients harboring the rs671 variant. Post-translational modifications are known to control GLUT1 trafficking and degradation. For instance, phosphorylation at Ser226 by protein kinase C promotes membrane localization 42 . Ubiquitination represents another regulatory mechanism. TRIM21, a RING-type E3 ligase, has been implicated in the turnover of metabolic enzymes including FASN, PFK1, and G6PD 43 – 47 . While PP2Aα has been reported to inhibit TRIM21-mediated GLUT1 degradation through dephosphorylation 48 , the specific linkage type and ubiquitination site were previously unknown. Our study identifies K48-linked polyubiquitination at lysine 256 as the dominant modification and implicates ALDH2 as a molecular scaffold facilitating this process. Interestingly, ALDH2 is not confined to mitochondria—it may also localize to the cytosol and nucleus—suggesting broader regulatory potential across subcellular compartments. In summary, we define a novel tumor-suppressive function of ALDH2 in repressing glucose metabolism and tumor progression in HCC. ALDH2 deficiency, as modeled by the rs671 variant, disrupts TRIM21-mediated ubiquitination of GLUT1, promoting metabolic activation and tumorigenesis. Targeting the ALDH2–TRIM21–GLUT1 axis may offer a promising therapeutic avenue, particularly for genetically predisposed patient populations. Several questions remain. The structural basis of ALDH2–TRIM21–GLUT1 interactions requires further elucidation. It is also unclear whether this regulatory mechanism is conserved in other GLUT1-high tumors. Finally, although BAY-876 showed therapeutic efficacy in vivo , its pharmacokinetics, toxicity profile, and potential synergy with existing therapies warrant further investigation. Future studies should also explore the therapeutic feasibility of restoring ALDH2 function in metabolically reprogrammed tumors. Methods Animals All animal procedures were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences. Mice were housed in a temperature-controlled and pathogen-free environment with a 12-hour light/12-hour dark cycle. ALDH2-KO mice were a kind gift from Drs. Jun Ren and Aijun Sun (Zhongshan Hospital, Fudan University, Shanghai, China) 49 , 50 . Rs671-KI mice were a gift from Dr. Yong Cang (ShanghaiTech University, Shanghai, China) 8 . Genome-edited F0 ALDH2 −/− mice were backcrossed to C57BL/6J mice for two generations. Wild-type and ALDH2 −/− mice were derived from crossing ALDH2 +/− mice. WT and ALDH2*2 (rs671, homozygous) mice were obtained by breeding ALDH2*1/*2 mice. AKO, ALDH2 rs671, and their respective control mice were fed a standard diet (P1101F-25). Hepatocellular carcinoma (HCC) mouse models were generated by intraperitoneal injection of 50 µg/g body weight of diethylnitrosamine (DEN) on postnatal day 14. Mice were euthanized at 37 weeks of age, and the liver tumor burden was assessed by quantifying visible tumor volume and number. Xenograft mouse model Four-week-old male athymic nude mice were purchased from Shanghai Slack Laboratory Animal Corp. Ltd. Hepa1-6 cells (2 × 10⁶) were resuspended in a 1:1 mixture of DMEM and Matrigel (Corning, 356234) and injected subcutaneously into the mice. Tumor dimensions were assessed every other day, and tumor volume was calculated using the formula: volume (mm³) = 0.52 × length × width². Mice tracing experiment with 13 C 6 -glucose Mice were fasted for 12 hours prior to euthanasia. They were injected intravenously with 1 g/kg [U-13C₆]-glucose (CLM-1396-PK, Cambridge Isotope Laboratories) via the tail vein at 15-minute intervals over a 30-minute labeling period. Liver tissues were promptly harvested for metabolic profiling. Chemical inhibition of GLUT1 in mice models WT and AKO mice were injected with 50 µg/g DEN on postnatal day 14. At week 28, mice were stratified into two cohorts. One group was orally administered BAY-876 (Selleck, S8452) at 5 mg/kg daily from week 28 to week 32, while the control group received vehicle (0.5% CMC-Na). Tumor volume was monitored throughout the treatment period. Plasma and liver tissues were collected for further experimental analysis. For the xenograft model, mice were randomized into two groups when tumor volumes reached approximately 100 mm³. Mice were orally administered either vehicle or 4 mg/kg BAY-876 daily until the study endpoint. BAY-876 was dissolved in 0.5% CMC-Na (Selleck, S6703), which was prepared in sterile saline. Cell culture, transfection and drug treatments Cell lines were obtained from the CAS Cell Bank and cultured in DMEM (Gibco, C11995500BT) supplemented with 10% FBS (Gibco, 10099141) and 1% penicillin–streptomycin (Invitrogen, 15140-122) in a humidified incubator with 5% CO 2 at 37°C. Transfection was performed using Lipofectamine 3000 (Thermofisher, L3000015) and Liji sheng wu (AC04L011) according to the manufacturer’s instructions. For drug treatments, MG132 (S2619) and CQ (S6999) were purchased from Selleck. CHX (HY-12320) was purchased from MCE. Medium containing a final concentration were then used to replace the original medium and cells were cultured in the presence of drugs for the indicated times were described in detail in the figure legend. Metabolic flux experiments using [U- 13 C 6 ]-glucose and gas chromatography/ mass spectrometry The metabolic flux analysis using [U- 13 C 6] -glucose followed our previously published protocol 51 – 53 . For stable isotope labeling, cells were seeded at a density of approximately 5 × 10 6 cells per 10-cm dish. When cells reached ~ 70% confluence, the unlabeled medium was replaced with labeling medium for indicated time. The labeling medium consisted of low glucose DMEM (Gibco,11054–020: 1 g/L unlabeled glucose, no glutamine) with supplement of 1 g/L [U- 13 C 6 ]-glucose (CLM-1396-PK, Cambridge Isotope Laboratory), 10% (v/v) fetal bovine serum, 1 mM pyruvate, 2 mM unlabeled L-glutamine, and 1% (v/v) penicillin-streptomycin. For sample pretreatment, cell pellets and 100 µL of medium, were resuspended in 400 µl of pre-colded 50% methanol/50% water containing 100 µM Norvaline (internal standard). The mixture was then thoroughly mixed, and rapidly frozen in dry ice for 30 min. Subsequently, 300 µl of chloroform was added, vortexed for 30 s, and centrifuged at 12,000 rpm at 4°C for 10 min. The upper aqueous phase was collected, freeze-concentrated, and dried. For tissue samples pretreatment, 30 µg of tissue was added to 400 µl of pre-colded 50% methanol/50% water containing 100 µM Norvaline. The sample was then homogenized using magnetic beads, immediately placed on dry ice for 30 minutes, and subsequently processed in the same manner as the cell samples. For sample derivatization, 20 mg/ml O-Isobutylhydroxylamine Hydrochloride (sigma, CIAH987ED435) was prepared in pyridine to protect ketone moieties. Each sample tube was supplemented with 70 µl of keto-protecting reagent, was thoroughly mixed, and incubated in a metal bath at 85°C for 20 minutes. After cooling, 30 µl of the derivating agent BSMFT (N-tert-Butyldimethylsilyl-N-Methytrifluoro-acetamide, sigma, 394882) was added to each tube, vortexed and incubated in a metal bath at 85°C for 60 min. Centrifuge at 12,000 rpm for 10 minutes at 4°C, transfer the supernatant to a vial for GC/MS analysis. GC-MS analysis was performed as described previously 54 . A Shimadzu QP-2010 Ultra GC-MS was programmed with an injection temperature of 250°C injection split ratio 1/10 and injected with 1 µL of sample. The GC column used was a 30 m × 0.25 mm × 0.25 mm HP-5ms. GC-MS data were analysed to determine isotope labelling andquantities of metabolites and implemented in MATLAB 55 . Western blot Cells were incubated in cell lysis (25 mM Tris-HCL, 150 mM NaCL, containing protease inhibitor cocktail and 2% (w/v) n-dodecyl-b-D-maltoside (APExBIO, C4421) for 2 h at 4 ◦ C and tissues were homogenized in 500 µL tissue lysis buffer. The soluble fractions of cell lysates were isolated by centrifugation at 4°C, 12,000 g for 10 min. Protein concentrations were determined using a protein assay kit (Beyotime, P0010). The supernatants were boiled for 10 min at 37°C. Proteins were separated by SDS-PAGE and transferred onto 0.45 µM PVDF membranes (Millipore, IPFL00005). After extensive rinse with TBST, immunoreactive bands were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using a chemiluminescence (ECL) kit (Proteintech) through Tanon 5200 Chemiluminescent Imaging System. All western blots were analyzed with ImageJ for quantitative measurements. Antibodies The following antibodies were used for immunoblotting. Anti-ALDH2 (15310-1-AP, 1:1000), Anti-GAPDH (60004-1-Ig, 1:5000), Anti-HA-Tag (66006-2-Ig, 1:1000), Anti-VINCULIN (26520-1-AP, 1:1000), Anti-FLAG-Tag (20543-1-AP, 1:1000) were purchased from Proteintech. Anti-GLUT1 (12939S, 1:1000), and normal rabbit IgG (2729S) were purchased from Cell Signaling Technology. Anti-GLUT1 (ab115730, 1:1000), were purchased from Abcam. Anti-Ubiquitin (Sc-8017, 1:500), and V5-Tag (Sc-81594, 1:1000) were purchased from Santa Cruz. Immunoprecipitation experiments HEK293T cells were transfected with target tagged protein using Attracting Transfection reagent (AC04L011, Liji shengwu), incubated by IP cell lysis buffer (50 mM Tris-HCL, 400 mM NaCl, 0.8% Triton X-100, pH 7.5), and cleared with targeted beads overnight. For LPC-H12 hepatocytes and liver tissues, cell lysates were incubated with target primary antibodies overnight at 4℃. Then protein A/G beads (B23202, Bimake) were added to each group and incubated with protein suspension for 4h at 4℃. Beads were washed with a wash buffer (50 mM Tris-HCl, 400Mm NaCl and 0.8% Triton-X-100, pH 7.5) three to five times. Proteins were eluted in sample buffer at 95℃ for 10 min and separated by SDS-PAGE and transferred to PVDF membrane for immunoblotting. Detection of interacting proteins with GLUT1 by using iTRAQ proteomics After immunoprecipitation for GLUT1 in LPC-H12 cells, we separated total cell lysate by using 10% SDS-PAGE for silver stain (catalog P0017S, Beyotime). The gel was extracted, and the proteins were identified using iTRAQ proteomics by Novognen. Immunofluorescence assay On the day prior to the experiment, the clean coverslips were immersed in 75% ethanol and then air dried and placed at the base of a six-well plate on the ultra-clean bench under a UV lamp for 6–12 hours to ensure sterility. On the day of the experiment, cells were seeded into the sterilized six-well plate with a cell density of 30%-50% with uniform distribution. Once the cells adhere to the surface, the culture medium was discarded and gently rinsed once with pre-chilled 1×PBS. The cells were fixed with 1 mL pre-chilled 4% PFA at room temperature for 10–20 min. After discarding the fixative, 1 mL of 1×PBS were gently added and placed on a shaker for 5 min. This process was repeated three times. One mL of 1% Triton X-100 was added at room temperature and incubated for 10 min. After discarding the permeabilization solution, 1 mL pre-chilled 1×PBS were washed on a shaker for 5 min. After three washes, 1 mL of 1% BSA was added at room temperature and incubated for 30 min. After discarding the blocking solution, the corresponding primary antibody (diluted according to the instructions) was added and incubated at 4°C overnight. The next day, 1 mL pre-chilled 1×PBS was added on a shaker for 5 min. The corresponding secondary antibody was added and incubated at 37°C in the dark for 30 min. After washing with 1 mL pre-chilled 1×PBS on a shaker for 5 min and repeating this process three times, DAPI was added for nuclear staining and sealed with transparent nail polish. After drying, images were taken using a Zeiss LSM 780 or stored in the dark at 4°C. Plasma membrane and intracellular membrane fractionation. Plasma membrane (PM) and intracellular membrane (ICM) proteins were isolated using the PM/ICM Protein Extraction Kit (ab65400, Abcam) following the manufacturer’s protocol. Briefly, cells were washed twice with ice-cold PBS, then incubated with 2 ml of homogenization buffer on ice and collected using a cell scraper. The cell suspension was homogenized on ice using a Dounce homogenizer (30–50 strokes), followed by centrifugation at 700×g for 5 min at 4°C. The supernatant was transferred to fresh tubes and centrifuged at 10,000×g for 30 min at 4°C. The resulting pellet contained total membrane proteins, while the supernatant contained cytosolic proteins. The membrane pellet was resuspended in 200 µl of upper-phase solution and mixed with 200 µl of lower-phase solution. After incubation on ice for 5 min, samples were centrifuged at 1,000×g for 5 min at 4°C to separate the phases. The upper-phase (containing PM proteins) and lower-phase (containing ICM proteins) were collected into separate tubes. To increase yield, phase separation was repeated by adding fresh upper-phase solution to the lower-phase and vice versa. The corresponding phases from both rounds were pooled and diluted with 5 volumes of water, then centrifuged at 15,000×g for 30 min at 4°C. The resulting pellets containing enriched PM or ICM proteins were collected for immunoblot analysis. Glucose uptake by 2-NBDG Seed cells 24 h prior to the experiment. When the cells reach 70–80% confluence, starve the cells in HBSS (Gibco, 14025134) medium for 2 h. Aspirate the medium and incubate the cells with 2-NBDG (APExBIO, B6035) solution for 5 min (37°C, 5% CO₂). Terminate the reaction by adding 1 mL of pre-chilled PBS. After obtaining the cell pellet, resuspend the cells in pre-chilled PBS and wash twice to remove unbound 2-NBDG. Finally, resuspend the cells in 500 µL PBS for flow cytometry analysis. Glucose consumption and lactate production The D-Glucose Kit (#10716251035, R-Biopharm) and the L-Lactic Acid Kit (#10139084035, R-Biopharm) were used to detect the concentration of glucose and lactate in the culture medium according to the manufacturer’s instructions. In brief, collect the cell culture media from 0h and 6h time points and place them on ice. For the assay, aliquot 4 µL of each sample, standards, and blanks into a 96-well plate. Add 50 µL of solution 1 and 96 µL of double-distilled water to each well, incubate at room temperature for 10 minutes, and measure the absorbance at 340 nm using a microplate reader; record these values as A1. Subsequently, add 1 µL of suspension 2 to each well and incubate at 37°C for 30 minutes. Measure the absorbance at 340 nm again and record these values as A2. Finally, calculate the glucose concentration in the culture media based on the standard curve. Human clinical samples and ALDH2 rs671 genotyping Tumor tissues and matched adjacent non-tumor tissues from 50 hepatocellular carcinoma (HCC) patients were obtained from the Eastern Hepatobiliary Surgery Hospital (Shanghai, China) and cryopreserved. Frozen tumor tissues were primarily used for protein analysis by western blotting. As for genotyping, genomic DNA was extracted from frozen human tissue samples using a commercial tissue DNA extraction kit according to the manufacturer’s protocol. DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The extracted genomic DNA was submitted to Bioshine Biotechnology (Hangzhou, China) for Sanger sequencing to determine the genotype of the ALDH2 rs671 polymorphism (c.1510G > A, Glu504Lys). Sequencing chromatograms were analyzed using SnapGene Viewer, and genotypes were classified as wild type (G/G), heterozygous (G/A), or homozygous mutant (A/A) based on the nucleotide at position c.1510 of the ALDH2 reference sequence (NM_000690.2). Cell proliferation assay Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, C0005, TargetMol) according to the manufacturer’s instructions. For pharmacological inhibition studies, cells were treated with DMSO or BAY-876 (5, 10, 20, or 40 nM; Selleck, S8452) for up to 96 hours. For gene silencing experiments, cells were transfected with siRNA targeting Slc2a1 (si-Slc2a1-1/2/3/4) or negative control siRNA (si-NC) using Lipofectamine 3000 for 24 hours prior to seeding. Cells were seeded at a density of 2 × 10³ cells per well in 96-well plates and allowed to adhere for 12 hours. Baseline absorbance (0 h) was measured following incubation with CCK-8 for 1 hour at 37°C, and absorbance at 450 nm was recorded using a microplate reader (BioTek). Cell viability was assessed every 24 hours by replacing the medium with fresh drug-containing medium supplemented with CCK-8. After 1 hour of incubation, absorbance at 450 nm was measured. Absorbance values were normalized to 0 h and background-subtracted using blank wells containing medium and CCK-8 without cells. Protein purification of GST-ALDH2/ GST-ALDH2 rs671, HA-TRIM21/HA-TRIM21ΔR and V5-GLUT1/ V5-GLUT1 K256R For bacterial expression, human ALDH2 and ALDH2 RS671 cDNAs were cloned into the pGEX vector to generate N-terminal GST-tagged fusion proteins. The constructs were transformed into E. coli BL21 (DE3) pLysS cells, cultured to an OD₆₀₀ of 0.8, and induced with 0.5 mM IPTG for 7 h at room temperature. Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 200 mM NaCl, 100 mM KCl, 20% glycerol, and 1 mM PMSF) after lysozyme treatment. Lysates were clarified by centrifugation, and recombinant proteins were purified by batch binding to GSTPur Glutathione beads. GST-tagged proteins were eluted with 15 mM reduced glutathione followed by overnight dialysis at 4°C to remove GSH. For mammalian expression, HEK293T cells were transfected at ~ 60% confluency with plasmids encoding HA-tagged TRIM21 or TRIM21ΔR, and V5-tagged GLUT1 or GLUT1 K256R, using Lipofectamine 3000. After 48 h, cells were lysed and recombinant proteins were purified using HA-tag or V5-tag affinity beads (P2185S, P2187S; Beyotime), following the manufacturer’s instructions. In vitro ubiquitination assay Ubiquitination reactions were performed in a 60 µL system containing 100 ng E1 (UBE1, 20433ES25, Yeasen), 500 ng E2 (UbcH5b/UBE2D2, HY-P79449, MCE), 500 ng purified E3 ligase (HA-TRIM21 or HA-TRIM21ΔR), 2 µg ubiquitin (20431ES08, Yeasen), 1 µg recombinant substrate protein (V5-GLUT1-His), and 1 µg GST or GST-ALDH2 as indicated. All reactions were carried out in ubiquitination buffer (Mg/ATP buffer, Yeasen) supplemented with 5 mM MgCl₂ and 2 mM ATP. After adding all components, the reaction mixtures were incubated at 30°C for 2 h. Reactions were terminated by adding 1× SDS sample loading buffer and incubating at 37°C for 10 min. Samples were then resolved by SDS–PAGE and analyzed by immunoblotting using anti-Ubiquitin antibodies to detect ubiquitinated forms of GLUT1. Untargeted Metabolomics Analysis Using UHPLC–QTOF-MS Untargeted metabolomics was performed as previously described 21 . Briefly, approximately 20 mg of liver tissue was collected and homogenized in 200 µL of HPLC-grade water using a pre-cooled tissue grinder (60 Hz, 2 minutes). A 10 µL aliquot of the homogenate was reserved for protein concentration measurement for downstream normalization. Quality control (QC) samples were prepared by pooling 10 µL aliquots from each individual homogenate. After homogenization, 800 µL of methanol/acetonitrile (1:1, v/v) containing internal standards was added to each sample, followed by vigorous vortexing for 30 seconds. The mixture was then subjected to ultrasonic homogenization on ice for 10 minutes, followed by snap-freezing in liquid nitrogen for 1 minute. This freeze–thaw cycle was repeated three times to ensure thorough metabolite extraction. Samples were subsequently stored at − 80°C for 1 hour to facilitate protein precipitation. Following centrifugation at 14,000 × g for 15 minutes at 4°C, the supernatant was transferred to fresh 1.5 mL tubes, and the solvent was evaporated to dryness under a gentle stream of nitrogen using a nitrogen evaporator. The dried residues were reconstituted in 100 µL of acetonitrile/water (1:1, v/v), centrifuged again at 13,000 × g for 15 minutes at 4°C, and 80 µL of the supernatant was transferred into LC–MS vials for analysis. Metabolomic profiling was carried out using an ultra-high-performance liquid chromatography (UHPLC) system coupled with a quadrupole time-of-flight (QTOF) mass spectrometer (TripleTOF 6600, SCIEX) operated in both positive and negative ion modes. Chromatographic separation was achieved using a Waters ACQUITY UPLC BEH Amide column (1.7 µm, 100 mm × 2.1 mm). The mobile phase composition, gradient program, and flow rate were applied as previously reported. All samples were injected in a random order. Blank samples (100% acetonitrile) and pooled QC samples were run every eight injections to monitor LC–MS system stability throughout the acquisition. Ethical All procedures performed in this study were approved by the Animal Ethics Committee of the SINH, CAS, Shanghai, China. and the Ethics Committee of the Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, China. Statistics Data is expressed as mean ± SD. Statistical analysis was conducted using the unpaired two-tailed Student’s t test for two groups by using Graph Pad Prism 9.0. And one-way ANOVA analysis for more than 2 groups. Differences were considered significant at a p value < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001. Study approval All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, China. All human samples were collected by Department of Hepatic Surgery I (Ward l), Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, China. Abbreviations ALDH2 aldehyde dehydrogenase 2 HCC hepatocellular carcinoma AKO ALDH2 knockout GLUT1 Glucose Transporter 1 SNP single nucleotide polymorphism DEN diethyl nitrosamine 2-NBDG 2-NBDG-6-phosphate WT wild-type TCA tricarboxylic acid CHX cycloheximide CQ chloroquine epiWAT epididymal white adipose tissue subWAT subcutaneous white adipose tissue. Declarations Acknowledgements This research was funded by grants from Shenzhen Medical Research Fund (SMRF B2302042), and National Natural Science Foundation of China (32030053), and RGC General Research Fund (9043653), Startup funds from the City University of Hong Kong (9380154), TBSC Project fund and Futian research project (9609327) and General Basic Research Program of Naval Military Medical University (No. 2023MS039). We are grateful to Prof. Dong Xie and Prof. Jingjing Li (SINH, China) for kindly providing the HA -TRIM21-WT and HA -TRIM21-ΔRING expression plasmids. We acknowledge the help from molecular biology core laboratory, animal facilities, and mass spectrometry facilities at Shanghai Institutes of Nutrition and Health (SINH), CAS, Shanghai. Author contributions H.Y.Y., S.S.Z., and L.L.Z designed this study. L.L.Z. and Y.Q.W performed most of the experiments and data analysis. N.N.L., R.L. performed liquid mass spectrometry analyses. Y.Z.T performed gas mass spectrometry analysis. C.Z.Y. and N.L. provided clinical HCC sample and analysis. X.C., X.Y., X.D.X., L.X.L., Q.C.T., J.W.L., H.M.J., H.R.M., C.X.D., S.T.C. helped with animal study and data analysis. L.L.Z., H.Y.Y. and S.S.Z drafted and reviewed this manuscript. Competing interests. The authors declare no competing interests. Materials & Correspondence. Huiyong Yin, Ph.D. Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, China 200031; Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China. Phone: (852) 3442-2812. E-mail: [email protected] Shanshan Zhong, Ph.D. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China. E-mail: [email protected] References Vogel A, Meyer T, Sapisochin G, Salem R, Saborowski A (2022) Hepatocellular carcinoma. Lancet 400:1345–1362 Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A et al (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71:209–249 Gross ER, Zambelli VO, Small BA, Ferreira JC, Chen CH, Mochly-Rosen D (2015) A personalized medicine approach for Asian Americans with the aldehyde dehydrogenase 2*2 variant. Annu Rev Pharmacol Toxicol 55:107–127 Chen CH, Ferreira JC, Gross ER, Mochly-Rosen D (2014) Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev 94:1–34 Brooks PJ, Enoch M-A, Goldman D, Li T-K, Yokoyama A (2009) The Alcohol Flushing Response: An Unrecognized Risk Factor for Esophageal Cancer from Alcohol Consumption. PLoS Med ;6 Matsuo K, Hamajima N, Shinoda M, Hatooka S, Inoue M, Takezaki T et al (2001) Gene-environment interaction between an aldehyde dehydrogenase-2 (ALDH2) polymorphism and alcohol consumption for the risk of esophageal cancer Carcinogenesis ;22:913–916 Farres J, Wang X, Takahashi K, Cunningham SJ, Wang TT, Weiner H (1994) Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase. J Biol Chem 269:13854–13860 Jin S, Chen J, Chen L, Histen G, Lin Z, Gross S et al (2015) ALDH2(E487K) mutation increases protein turnover and promotes murine hepatocarcinogenesis. Proceedings of the National Academy of Sciences ;112:9088–9093 Gao Y, Zhou Z, Ren T, Kim SJ, He Y, Seo W et al (2019) Alcohol inhibits T-cell glucose metabolism and hepatitis in ALDH2-deficient mice and humans: roles of acetaldehyde and glucocorticoids. Gut 68:1311–1322 Jarl J, Gerdtham UG (2012) Time pattern of reduction in risk of oesophageal cancer following alcohol cessation–a meta-analysis. Addiction 107:1234–1243 Tomoda T, Nouso K, Sakai A, Ouchida M, Kobayashi S, Miyahara K et al (2012) Genetic risk of hepatocellular carcinoma in patients with hepatitis C virus: A case control study. J Gastroenterol Hepatol 27:797–804 Susumu Higuchi SM, Taro, Muramatsu (1996) Masanobu Murayama, and Motoi Hayashida. Alcohol and Aldehyde Dehydrogenase Genotypes and Drinking Behavior in Japanese. Alcohol Clin Experimental Res Pautassi RM, Camarini R, Quadros IM, Miczek KA, Israel Y (2010) Genetic and Environmental Influences on Ethanol Consumption: Perspectives From Preclinical Research. Alcoholism: Clin Experimental Res 34:976–987 Zhong S, Li L, Liang N, Zhang L, Xu X, Chen S et al (2021) Acetaldehyde Dehydrogenase 2 regulates HMG-CoA reductase stability and cholesterol synthesis in the liver. Redox Biol 41:101919 Li L, Zhong S, Li R, Liang N, Zhang L, Xia S et al (2022) Aldehyde dehydrogenase 2 and PARP1 interaction modulates hepatic HDL biogenesis by LXRalpha-mediated ABCA1 expression. JCI Insight ;7 Hanahan D (2022) Hallmarks of Cancer: New Dimensions. Cancer Discov 12:31–46 Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324:1029–1033 Czernin J, Phelps ME (2002) Positron emission tomography scanning: current and future applications. Annu Rev Med 53:89–112 Joost H-G, Bell GI, Best JD, Birnbaum MJ, Charron MJ, Chen YT et al (2002) Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators. Am J Physiology-Endocrinology Metabolism 282:E974–E976 Amann T, Maegdefrau U, Hartmann A, Agaimy A, Marienhagen J, Weiss TS et al (2009) GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis. Am J Pathol 174:1544–1552 Shen X, Wang C, Liang N, Liu Z, Li X, Zhu ZJ et al (2021) Serum Metabolomics Identifies Dysregulated Pathways and Potential Metabolic Biomarkers for Hyperuricemia and Gout. Arthritis Rheumatol 73:1738–1748 Karim S (2012) Hepatic expression and cellular distribution of the glucose transporter family. World J Gastroenterol ;18 Szablewski L (2013) Expression of glucose transporters in cancers. Biochim et Biophys Acta (BBA) - Reviews Cancer 1835:164–169 Adekola K, Rosen ST, Shanmugam M (2012) Glucose transporters in cancer metabolism. Curr Opin Oncol 24:650–654 Mueckler M, Thorens B (2013) The SLC2 (GLUT) family of membrane transporters. Mol Aspects Med 34:121–138 Siebeneicher H, Cleve A, Rehwinkel H, Neuhaus R, Heisler I, Müller T et al (2016) Identification and Optimization of the First Highly Selective GLUT1 Inhibitor BAY-876. ChemMedChem 11:2261–2271 Zhang J, Zhao X, Guo Y, Liu Z, Wei S, Yuan Q et al (2022) Macrophage ALDH2 (Aldehyde Dehydrogenase 2) Stabilizing Rac2 Is Required for Efferocytosis Internalization and Reduction of Atherosclerosis Development. Arterioscler Thromb Vasc Biol 42:700–716 Cilenti L, Di Gregorio J, Ambivero CT, Andl T, Liao R, Zervos AS (2020) Mitochondrial MUL1 E3 ubiquitin ligase regulates Hypoxia Inducible Factor (HIF-1α) and metabolic reprogramming by modulating the UBXN7 cofactor protein. Sci Rep ;10 Chen X, Cao M, Wang P, Chu S, Li M, Hou P et al (2022) The emerging roles of TRIM21 in coordinating cancer metabolism, immunity and cancer treatment. Front Immunol 13:968755 Swatek KN, Komander D (2016) Ubiquitin modifications. Cell Res 26:399–422 Siegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer statistics, 2023. CA Cancer J Clin 73:17–48 Hou G, Chen L, Liu G, Li L, Yang Y, Yan HX et al (2017) Aldehyde dehydrogenase-2 (ALDH2) opposes hepatocellular carcinoma progression by regulating AMP‐activated protein kinase signaling in mice. Hepatology 65:1628–1644 Boroughs LK, DeBerardinis RJ (2015) Metabolic pathways promoting cancer cell survival and growth. Nat Cell Biol 17:351–359 Wohlfart DP, Lou B, Middel CS, Morgenstern J, Fleming T, Sticht C et al (2022) Accumulation of acetaldehyde in aldh2.1(-/-) zebrafish causes increased retinal angiogenesis and impaired glucose metabolism. Redox Biol 50:102249 Ma LL, Ding ZW, Yin PP, Wu J, Hu K, Sun AJ et al (2021) Hypertrophic preconditioning cardioprotection after myocardial ischaemia/reperfusion injury involves ALDH2-dependent metabolism modulation. Redox Biol 43:101960 Cao R, Fang D, Wang J, Yu Y, Ye H, Kang P et al (2019) ALDH2 Overexpression Alleviates High Glucose-Induced Cardiotoxicity by Inhibiting NLRP3 Inflammasome Activation. J Diabetes Res 2019:4857921 Wang C, Fan F, Cao Q, Shen C, Zhu H, Wang P et al (2016) Mitochondrial aldehyde dehydrogenase 2 deficiency aggravates energy metabolism disturbance and diastolic dysfunction in diabetic mice. J Mol Med (Berl) 94:1229–1240 Amann T, Hellerbrand C (2009) GLUT1 as a therapeutic target in hepatocellular carcinoma. Expert Opin Ther Targets 13:1411–1427 Osthus RC, Shim H, Kim S, Li Q, Reddy R, Mukherjee M et al (2000) Deregulation of glucose transporter 1 and glycolytic gene expression by c-Myc. J Biol Chem 275:21797–21800 Zhang C, Liu Z, Wang F, Zhang B, Zhang X, Guo P et al (2023) Nanomicelles for GLUT1-targeting hepatocellular carcinoma therapy based on NADPH depletion. Drug Deliv 30:2162160 Cohen NR, Knecht DA, Lodish HF (1996) Functional expression of rat GLUT 1 glucose transporter in Dictyostelium discoideum. Biochem J 315:971–975 Lee EE, Ma J, Sacharidou A, Mi W, Salato VK, Nguyen N et al (2015) A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome. Mol Cell 58:845–853 Zheng N, Shabek N (2017) Ubiquitin Ligases: Structure, Function, and Regulation. Annu Rev Biochem 86:129–157 Ozato K, Shin DM, Chang TH, Morse HC (2008) 3rd. TRIM family proteins and their emerging roles in innate immunity. Nat Rev Immunol 8:849–860 Gu L, Zhu Y, Lin X, Tan X, Lu B, Li Y (2020) Stabilization of FASN by ACAT1-mediated GNPAT acetylation promotes lipid metabolism and hepatocarcinogenesis. Oncogene 39:2437–2449 Cheng J, Huang Y, Zhang X, Yu Y, Wu S, Jiao J et al (2020) TRIM21 and PHLDA3 negatively regulate the crosstalk between the PI3K/AKT pathway and PPP metabolism. Nat Commun 11:1880 Lee JH, Liu R, Li J, Zhang C, Wang Y, Cai Q et al (2017) Stabilization of phosphofructokinase 1 platelet isoform by AKT promotes tumorigenesis. Nat Commun 8:949 Gu M, Tan M, Zhou L, Sun X, Lu Q, Wang M et al (2022) Protein phosphatase 2Acα modulates fatty acid oxidation and glycolysis to determine tubular cell fate and kidney injury. Kidney Int 102:321–336 Sun A, Ren J (2013) ALDH2, a novel protector against stroke? Cell Res 23:874–875 Ma H, Guo R, Yu L, Zhang Y, Ren J (2011) Aldehyde dehydrogenase 2 (ALDH2) rescues myocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic aldehyde. Eur Heart J 32:1025–1038 Li M, He X, Guo W, Yu H, Zhang S, Wang N et al (2020) Aldolase B suppresses hepatocellular carcinogenesis by inhibiting G6PD and pentose phosphate pathways. Nat Cancer 1:735–747 Wang N, Liu H, Liu G, Li M, He X, Yin C et al (2020) Yeast beta-D-glucan exerts antitumour activity in liver cancer through impairing autophagy and lysosomal function, promoting reactive oxygen species production and apoptosis. Redox Biol 32:101495 Liu G, Shi A, Wang N, Li M, He X, Yin C et al (2020) Polyphenolic Proanthocyanidin-B2 suppresses proliferation of liver cancer cells and hepatocellular carcinogenesis through directly binding and inhibiting AKT activity. Redox Biol 37:101701 Ma L, Tao Y, Duran A, Llado V, Galvez A, Barger JF et al (2013) Control of nutrient stress-induced metabolic reprogramming by PKCzeta in tumorigenesis. Cell 152:599–611 Portnoy VA, Scott DA, Lewis NE, Tarasova Y, Osterman AL, Palsson BO (2010) Deletion of genes encoding cytochrome oxidases and quinol monooxygenase blocks the aerobic-anaerobic shift in Escherichia coli K-12 MG1655. Appl Environ Microbiol 76:6529–6540 Additional Declarations There is NO Competing Interest. Supplementary Files 20250709supplementalfigures.docx supplementary figures GA.png Graphical abstract Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7102103","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":497486803,"identity":"237e7ed3-343f-46d9-877f-2ac82965af88","order_by":0,"name":"Huiyong Yin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACAwbGBiAlwcAPYQDBAQY2GBO/FskGxsYGIrXAGAdg1hDSYi6R3Cbxsc0iz/j44fbHvG0Mcnw3EtgezsCjxXJGYpvkzDaJYrMziY3NQC3GkjcS2A034HPYjcQ2ad42icRtNxjBWhI3AG2RfECMls0zIFrqideyQQKiJcEApAWvw848bLaccU4icQbQLzPnnJMwnHnmYZskPu8bHE9/eONDWV1if/vxBx/elNnI8x1PPibZg0cLELBIMLJBWEw8wDhlYMAbK2DA/IHhD4TF+IOQ2lEwCkbBKBiRAAAWjlSxDvK5IgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-7049-1560","institution":"City University of Hong Kong","correspondingAuthor":true,"prefix":"","firstName":"Huiyong","middleName":"","lastName":"Yin","suffix":""},{"id":497486804,"identity":"ffba7ae7-aa1d-43ba-9064-1b2672a4b2af","order_by":1,"name":"Shanshan Zhong","email":"","orcid":"","institution":"City University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Zhong","suffix":""},{"id":497486805,"identity":"ded87189-9cf5-4bd8-89dc-e50fe71d4ff8","order_by":2,"name":"Lili Zhang","email":"","orcid":"","institution":"CAS Key Laboratory of Nutrition Metabolism and Food Safety Shanghai Institutes of Nutrition and Health, Chinese Academy of Sciences (CAS), Shanghai, China, and University of Chinese Academy of Scien","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Zhang","suffix":""},{"id":497486806,"identity":"567495bd-feed-4f87-add1-feef6dff7153","order_by":3,"name":"Yongqiang Wang","email":"","orcid":"","institution":"CAS Key Laboratory of Nutrition Metabolism and Food Safety Shanghai Institutes of Nutrition and Health, Chinese Academy of Sciences (CAS), Shanghai, China, and University of Chinese Academy of Scien","correspondingAuthor":false,"prefix":"","firstName":"Yongqiang","middleName":"","lastName":"Wang","suffix":""},{"id":497486807,"identity":"1fa2a4ce-4958-46d3-8639-23c6d5e6a807","order_by":4,"name":"Ningning Liang","email":"","orcid":"","institution":"CAS Key Laboratory of Nutrition Metabolism and Food Safety Shanghai Institutes of Nutrition and Health, Chinese Academy of Sciences (CAS), Shanghai, China, and University of Chinese Academy of Scien","correspondingAuthor":false,"prefix":"","firstName":"Ningning","middleName":"","lastName":"Liang","suffix":""},{"id":497486808,"identity":"252c6dc5-7db6-4046-ae4b-ee96bc693c10","order_by":5,"name":"Xin Chen","email":"","orcid":"","institution":"Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Chen","suffix":""},{"id":497486809,"identity":"2bc5eaba-ed79-427f-bfdf-bf7f4a9a6d34","order_by":6,"name":"Xuan Yuan","email":"","orcid":"","institution":"Institute of Metabolic Diseases, Qingdao University, Qingdao, China; Shandong Provincial Key Laboratory of Metabolic Diseases, Qingdao Key Laboratory of Gout, the Affiliated Hospital of Qingdao Univ","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Yuan","suffix":""},{"id":497486810,"identity":"ef5f8c0f-4488-4ff2-b4d2-12e3f4aacd31","order_by":7,"name":"Rui Li","email":"","orcid":"","institution":"ShanghaiTech University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Li","suffix":""},{"id":497486811,"identity":"b0dfd198-3d1e-4fa2-a8b7-30ff95f47da5","order_by":8,"name":"Chunzhao Yin","email":"","orcid":"","institution":"ShanghaiTech University","correspondingAuthor":false,"prefix":"","firstName":"Chunzhao","middleName":"","lastName":"Yin","suffix":""},{"id":497486812,"identity":"941ab72d-8e30-4cae-8d4a-858647d20ae7","order_by":9,"name":"Chenxi Du","email":"","orcid":"","institution":"ShanghaiTech University","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Du","suffix":""},{"id":497486813,"identity":"0a7665ed-a29f-4b7c-9a4a-5f6e75775f08","order_by":10,"name":"Xiaodong Xu","email":"","orcid":"","institution":"CAS Key Laboratory of Nutrition Metabolism and Food Safety Shanghai Institutes of Nutrition and Health, Chinese Academy of Sciences (CAS), Shanghai, China, and University of Chinese Academy of Scien","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Xu","suffix":""},{"id":497486814,"identity":"f3b71b3b-ec19-4211-8469-4e0bc752df24","order_by":11,"name":"Luxiao Li","email":"","orcid":"","institution":"CAS Key Laboratory of Nutrition Metabolism and Food Safety Shanghai Institutes of Nutrition and Health, Chinese Academy of Sciences (CAS), Shanghai, China, and University of Chinese Academy of Scien","correspondingAuthor":false,"prefix":"","firstName":"Luxiao","middleName":"","lastName":"Li","suffix":""},{"id":497486815,"identity":"442d4fd2-20e8-4c87-9530-7e4cf925670a","order_by":12,"name":"Qiaochu Tu","email":"","orcid":"","institution":"ShanghaiTech University","correspondingAuthor":false,"prefix":"","firstName":"Qiaochu","middleName":"","lastName":"Tu","suffix":""},{"id":497486816,"identity":"ddfae663-134e-409a-aac5-e062031b9b15","order_by":13,"name":"Jingwen Lv","email":"","orcid":"","institution":"CAS Key Laboratory of Nutrition Metabolism and Food Safety Shanghai Institutes of Nutrition and Health, Chinese Academy of Sciences (CAS), Shanghai, China, and University of Chinese Academy of Scien","correspondingAuthor":false,"prefix":"","firstName":"Jingwen","middleName":"","lastName":"Lv","suffix":""},{"id":497486817,"identity":"ec43cfad-739d-40bd-b0b9-2aa207310647","order_by":14,"name":"Huimin Jiang","email":"","orcid":"","institution":"CAS Key Laboratory of Nutrition Metabolism and Food Safety Shanghai Institutes of Nutrition and Health, Chinese Academy of Sciences (CAS), Shanghai, China, and University of Chinese Academy of Scien","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Jiang","suffix":""},{"id":497486818,"identity":"99ad1c45-6934-48f9-8d8c-1b0a39323fc0","order_by":15,"name":"Haoran Ma","email":"","orcid":"","institution":"CAS Key Laboratory of Nutrition Metabolism and Food Safety Shanghai Institutes of Nutrition and Health, Chinese Academy of Sciences (CAS), Shanghai, China, and University of Chinese Academy of Scien","correspondingAuthor":false,"prefix":"","firstName":"Haoran","middleName":"","lastName":"Ma","suffix":""},{"id":497486819,"identity":"22104669-b131-41bc-91be-80240ded9058","order_by":16,"name":"Shiting Chen","email":"","orcid":"","institution":"CAS Key Laboratory of Nutrition Metabolism and Food Safety Shanghai Institutes of Nutrition and Health, Chinese Academy of Sciences (CAS), Shanghai, China, and University of Chinese Academy of Scien","correspondingAuthor":false,"prefix":"","firstName":"Shiting","middleName":"","lastName":"Chen","suffix":""},{"id":497486820,"identity":"d9aa391b-6329-4d87-ae05-2542a10a3ec6","order_by":17,"name":"Yongzhen Tao","email":"","orcid":"https://orcid.org/0000-0003-1710-5346","institution":"CAS","correspondingAuthor":false,"prefix":"","firstName":"Yongzhen","middleName":"","lastName":"Tao","suffix":""},{"id":497486821,"identity":"af72e77d-0122-4ca8-a83b-bc073f16a924","order_by":18,"name":"Nan Li","email":"","orcid":"","institution":"Eastern Hepatobiliary Surgery Hospital","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-11 13:25:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7102103/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7102103/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88748900,"identity":"19955850-1e7a-483b-b6f9-9fc34031b201","added_by":"auto","created_at":"2025-08-11 05:31:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":453885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALDH2 is downregulated in HCC and its deficiency promotes tumorigenesis in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A–B)\u003c/strong\u003e ALDH2 mRNA expression levels in HCC tumor versus adjacent normal liver tissues from the TCGA-LIHC (tumor, n=373; normal, n=50, A) and GSE14520 cohorts (tumor, n = 224; normal, n=220, B). \u003cstrong\u003e(C)\u003c/strong\u003e Kaplan–Meier overall survival analysis of HCC patients in the TCGA cohort stratified by ALDH2 expression level (data obtained from GEPIA). \u003cstrong\u003e(D–E)\u003c/strong\u003e Immunoblot analysis of ALDH2 protein expression in 24 paired human HCC tumor (T) and adjacent non-tumor (N) liver tissues (D), and quantification of ALDH2 expression across all patient pairs (E).\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eF) \u003c/strong\u003eSchematic diagram of the DEN-induced HCC model. Wild-type (WT) and ALDH2 knockout (AKO) mice were injected with 50ug/g DEN at 2 weeks of age and sacrificed at 37\u003csup\u003eth\u003c/sup\u003e week (left). Representative liver images and H\u0026amp;E staining of tumor nodules are shown (right). Scale bars, 50μm; N, Normal; T, tumor. \u003cstrong\u003e(G-J)\u003c/strong\u003e Quantification of liver-to-body weight ratio (G), total number of visible surface tumors (H), maximum tumor volume(I) and tumor number distribution (J) in WT (n = 13) and AKO (n = 10) mice. Statistical comparisons were performed using two-tailed Student’s t test. All data are presented as mean ± SD.*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/ebe2e52cdbd63cfa4758d86f.png"},{"id":88748898,"identity":"de53fbd0-1d41-4240-90a2-1a81f2d426de","added_by":"auto","created_at":"2025-08-11 05:31:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":315874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolomic and isotope tracing reveal enhanced glucose flux with ALDH2 loss in hepatocellular carcinoma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eHeatmap showing the relative abundance of significantly altered metabolites in DEN-induced liver tissues from wild-type (WT, n = 5) and ALDH2 knockout (AKO, n = 5) mice. Metabolites are categorized by functional class.\u003cstrong\u003e (B) \u003c/strong\u003ePathway enrichment analysis of differentially abundant metabolites between WT and AKO livers, highlighting alterations in glucose metabolism–related pathways. \u003cstrong\u003e(C)\u003c/strong\u003e Schematic of [U-¹³C₆]-glucose tail vein injection and in vivo metabolic flux analysis. Mice were sacrificed 30 minutes post-injection for MS-based metabolite tracing.\u003cstrong\u003e (D-E)\u003c/strong\u003e Fractional enrichment of ¹³C-labeled pyruvate, lactate, and alanine in livers from WT and AKO (D), and WT and ALDH2 rs671 knock-in (E) mice following [U-¹³C₆]-glucose infusion.\u003cstrong\u003e (F)\u003c/strong\u003e Flow cytometry analysis of glucose uptake in sh-ALDH2 and control (sh-NC) LPC-H12 cells treated with 100 µM 2-NBDG for 5 minutes. \u003cstrong\u003e(G)\u003c/strong\u003e Quantification of extracellular glucose consumption in sh-ALDH2 and sh-NC cells, measured using a glucose assay kit and normalized to total protein concentration. Statistical comparisons were performed using two-tailed Student’s t test. All data are presented as mean ± SD.*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/ce636ecbcea48b10d509204e.png"},{"id":88749724,"identity":"859837c0-5d97-4233-977e-e4b986a6d865","added_by":"auto","created_at":"2025-08-11 05:39:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":501882,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALDH2 deficiency promotes GLUT1-dependent glucose metabolism, which can be suppressed by GLUT1 inhibitor BAY-876.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eImmunoblot analysis of GLUT1 expression in DEN-induced liver tumors from wild-type (WT), ALDH2 knockout (AKO), and ALDH2 rs671 knock-in (rs671) mice. \u003cstrong\u003e(B)\u003c/strong\u003e GLUT1 protein levels in LPC-H12 and Hepa1-6 HCC cells stably expressing shRNA against ALDH2 (sh-ALDH2) or a non-targeting control (sh-NC). \u003cstrong\u003e(C)\u003c/strong\u003e Immunoblot analysis of GLUT1 and ALDH2 expression in 12 paired human HCC tumor (T) and adjacent non-tumor (N) tissues. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of GLUT1 expression in tumor tissues from HCC patients stratified by ALDH2 genotype (n = 23 for WT, n = 27 for rs671). \u003cstrong\u003e(E) \u003c/strong\u003eCorrelation between ALDH2 and GLUT1 protein levels across 50 primary HCC tumors. Pearson correlation was used for statistical analysis.\u003cstrong\u003e (F)\u003c/strong\u003e Subcellular fractionation analysis of GLUT1 in intracellular membrane (ICM) and plasma membrane (PM) compartments from liver tissues of WT and AKO mice. ATP1A1 and GAPDH served as PM and cytosolic markers, respectively; WCL, whole cell lysate.\u003cstrong\u003e (G)\u003c/strong\u003e Quantification of GLUT1 distribution in subcellular fractions. \u003cstrong\u003e(H)\u003c/strong\u003e Glucose consumption in sh-ALDH2 and control cells treated with or without 50 nM BAY-876, measured using a glucose assay kit and normalized to total protein.\u003cstrong\u003e (I)\u003c/strong\u003e Fractional labeling of glycolytic and TCA cycle metabolites in LPC-H12 cells treated with [U-¹³C₆]-glucose and either BAY-876 (50 nM) or DMSO for 6 h.\u003cstrong\u003e (J)\u003c/strong\u003e Lactate concentration in the culture medium of sh-ALDH2 and control cells treated with or without BAY-876 (50 nM) treatment.\u003cstrong\u003e (K) \u003c/strong\u003eKaplan–Meier overall survival of HCC patients (n = 362) stratified by combined ALDH2 and GLUT1 expression levels. Data obtained from the TCGA-LIHC cohort; survival analysis performed using cSurvival. Statistical comparisons were performed using two-tailed Student’s t test (D, G) or one-way ANOVA (H-K). All data are presented as mean ± SD.\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/aa6e7c18126fd40e3c60e3ac.png"},{"id":88749723,"identity":"4f145f58-24f4-47c8-8447-7696b1c5a14f","added_by":"auto","created_at":"2025-08-11 05:39:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":576770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALDH2 interacts with GLUT1 and promotes its proteasomal degradation via ubiquitination. (A) \u003c/strong\u003eImmunoblot analysis of GLUT1 protein levels in LPC-H12 cells treated with cycloheximide (CHX, 50 μg/mL) for the indicated time points, with or without stable ALDH2 knockdown.\u003cstrong\u003e (B) \u003c/strong\u003eSemi-quantification of GLUT1 protein levels following ALDH2 knockdown.\u003cstrong\u003e (C) \u003c/strong\u003eRepresentative immunoblot showing GLUT1 levels in LPC-H12 cells treated with CHX in the presence or absence of the proteasome inhibitor MG132 (10 μM) or the lysosome inhibitor chloroquine (CQ, 20 μM) for the indicated time. \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of GLUT1 abundance following treatment. \u003cstrong\u003e(E-F) \u003c/strong\u003eEndogenous ubiquitination of GLUT1 in liver tissues from WT and ALDH2 knockout (AKO) mice (E), and WT and rs671 knock-in mice (F), assessed by immunoprecipitation (IP) with anti-GLUT1 antibody followed by immunoblotting with anti-ubiquitin.\u003cstrong\u003e (G) \u003c/strong\u003eIn vivo ubiquitination assay in 293T cells co-transfected with V5-tagged GLUT1, HA-tagged ubiquitin, and either Flag-tagged WT or rs671 ALDH2. Cells were treated with MG132 (10 μM) for 6 hours prior to IP.\u003cstrong\u003e (H-I) \u003c/strong\u003eCo-IP of endogenous GLUT1 and ALDH2 in liver tissues from WT and AKO mice (H), and WT and rs671 mice (I).\u003cstrong\u003e(J) \u003c/strong\u003eCo-IP analysis of endogenous ALDH2–GLUT1 interaction in paired human HCC tumor (T) and adjacent non-tumor (N) tissues.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/65453dabfb756bd0164fb378.png"},{"id":88749725,"identity":"5df9e851-226d-4a1f-9363-6dc5b1737a3c","added_by":"auto","created_at":"2025-08-11 05:39:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":772085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALDH2 interacts with TRIM21 and catalyzes K48-linked ubiquitination of GLUT1. (A)\u003c/strong\u003e Proteomic identification of TRIM21 and MUL1 as candidate ALDH2- and GLUT1-associated E3 ligases. Protein lysates were immunoprecipitated with anti-ALDH2 or anti-GLUT1, and shared interacting proteins were analyzed by mass spectrometry.\u003cstrong\u003e (B)\u003c/strong\u003e Immunoblot analysis of GLUT1 protein levels in LPC-H12 cells following siRNA-mediated knockdown of MUL1 or TRIM21, with or without ALDH2 knockdown. \u003cstrong\u003e(C) \u003c/strong\u003eUbiquitination assay in 293T cells transfected with V5-tagged GLUT1, HA-tagged TRIM21, FLAG-tagged MUL1, and HA-ubiquitin. Cells were treated with MG132 (10 μM, 6 h) prior to harvest.\u003cstrong\u003e (D) \u003c/strong\u003eUbiquitination assay in 293T cells expressing wild-type or ΔRING mutant TRIM21 to assess the requirement of E3 ligase activity. Cells were treated with MG132 (10 μM, 6 h)\u003cstrong\u003e. (E)\u003c/strong\u003e Co-IP of endogenous TRIM21 with ALDH2 and GLUT1 in liver tissues from wild-type (WT), ALDH2 knockout (AKO), and rs671 knock-in mice. \u003cstrong\u003e(F)\u003c/strong\u003e Co-IP of TRIM21 with ALDH2 and GLUT1 in three paired human HCC and adjacent non-tumor tissues. \u003cstrong\u003e(G)\u003c/strong\u003e Quantification of TRIM21-associated ALDH2 and GLUT1 levels in non-tumor (N) and tumor (T) tissues shown in (F).\u003cstrong\u003e (H) \u003c/strong\u003eIn vitro ubiquitination assay testing the requirement of TRIM21 E3 ligase activity. Reactions included V5-GLUT1, wild-type or ΔRING TRIM21, GST-ALDH2, E1/E2 enzymes, ATP, and ubiquitin. \u003cstrong\u003e(I) \u003c/strong\u003eIn vitro ubiquitination assay testing ALDH2 dose-dependency in TRIM21-mediated GLUT1 ubiquitination. Reactions included increasing amounts of GST-ALDH2 with fixed TRIM21 and V5-GLUT1.\u003cstrong\u003e (J) \u003c/strong\u003eUbiquitination assays in 293T cells co-expressing HA-tagged K48 or K63 ubiquitin, V5-GLUT1, and HA-TRIM21 to identify ubiquitin linkage specificity. \u003cstrong\u003e(K) \u003c/strong\u003eUbiquitination assays assessing the effect of wild-type versus rs671 ALDH2 on TRIM21-mediated K48-linked GLUT1 ubiquitination. Cells were co-transfected with HA-K48-Ub, V5-GLUT1, HA-TRIM21, and either FLAG-tagged WT or rs671 ALDH2\u003cstrong\u003e. \u003c/strong\u003eStatistical analyses were performed using two-tailed Student’s t test. All data are presented as mean ± SD.*p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/1d43bfe20dfa639a7243c575.png"},{"id":88749915,"identity":"ab0b8655-d2c9-4c0a-8897-2b084a21ce23","added_by":"auto","created_at":"2025-08-11 05:47:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":531970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALDH2 promotes ubiquitination of GLUT1 at lysine 256 (K256), which is critical for GLUT1 degradation and tumor suppression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eMass spectrometry (MS) analysis identifying lysine 256 (K256) as a potential ubiquitination site on GLUT1 in LPC-H12 cells. \u003cstrong\u003e(B) \u003c/strong\u003eA multiple sequence alignment of GLUT1 orthologs across species, highlighting the conserved K256 residue in red.\u003cstrong\u003e (C)\u003c/strong\u003e Western blot analysis of GLUT1 protein stability in 293T cells expressing either wild-type (WT) or K256R-mutant GLUT1 following cycloheximide (CHX, 50 μg/mL) treatment for the indicated durations.\u003cstrong\u003e (D) \u003c/strong\u003eUbiquitination assays in 293T cells co-transfected with V5-tagged GLUT1^WT or GLUT1^K256R, HA-tagged TRIM21, and HA-ubiquitin. Cells were treated with MG132 (10 μM, 6 h) prior to harvesting.\u003cstrong\u003e (E) \u003c/strong\u003eIn vitro ubiquitination assays using purified V5-GLUT1^WT or K256R, HA-tagged TRIM21, GST-tagged ALDH2, E1/E2 enzymes, ATP, and ubiquitin. \u003cstrong\u003e(F) \u003c/strong\u003eSchematic of subcutaneous xenograft experiments using Hep1-6 cells stably expressing GLUT1^WT or GLUT1^K256R, with or without ALDH2 overexpression.\u003cstrong\u003e (G) \u003c/strong\u003eImmunoblot analysis confirming GLUT1 expression in harvested tumor tissues.\u003cstrong\u003e (H) \u003c/strong\u003eRepresentative images of xenograft tumors (n = 6 per group). \u003cstrong\u003e(I-J) \u003c/strong\u003eTumor volume and final tumor weight measured at the endpoint.\u003cstrong\u003e \u003c/strong\u003eData are presented as mean ± SD. Statistical significance was determined using one-way ANOVA. ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/26119ed1ac6b3ad810c03cf6.png"},{"id":88749916,"identity":"cd6f2725-0823-4821-9f2a-0493ff03410f","added_by":"auto","created_at":"2025-08-11 05:47:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":380424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLUT1 inhibition or knockdown attenuates ALDH2 deficiency-induced tumorigenesis in nude mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic illustration of the xenograft experimental design. Hepa1-6 cells stably expressing control shRNA (sh-NC) or ALDH2 shRNA (sh-ALDH2) were subcutaneously injected into the flanks of nude mice, followed by daily oral administration of vehicle or the selective GLUT1 inhibitor BAY-876 (4 mg/kg). \u003cstrong\u003e(B-D) \u003c/strong\u003eTumor volume and tumor weight were recorded in each group (n=6).\u003cstrong\u003e (E) \u003c/strong\u003eSchematic representation of the second xenograft model. Hepa1-6 cells with dual knockdown combinations (sh-NC–sh-NC, sh-ALDH2–sh-NC, sh-ALDH2–sh-GLUT1-1, and sh-ALDH2–sh-GLUT1-4) were injected subcutaneously into nude mice.\u003cstrong\u003e (F) \u003c/strong\u003eWestern blot analysis confirming GLUT1 knockdown in tumor tissues.\u003cstrong\u003e (G-I) \u003c/strong\u003eTumor volume and tumor weight were assessed in each group (n = 6).\u003cstrong\u003e \u003c/strong\u003eStatistical analyses were performed using one-way ANOVA. All data are presented as mean ± SD.*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/0ec132ef4ac06697b90cbad8.png"},{"id":88748906,"identity":"26841f57-7897-45e6-beab-3a4a1c62018f","added_by":"auto","created_at":"2025-08-11 05:31:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":132903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical summary of the ALDH2–TRIM21–GLUT1 axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn normal hepatocytes (left), ALDH2 facilitates the recruitment of the E3 ubiquitin ligase TRIM21 to GLUT1, promoting K48-linked polyubiquitination at lysine 256 and subsequent proteasomal degradation of GLUT1. This regulatory axis limits glucose uptake and glycolysis, thereby constraining tumor growth.\u003c/p\u003e\n\u003cp\u003eIn contrast, ALDH2 deficiency (right) in ALDH2 rs 671 or KO, disrupts the TRIM21–GLUT1 interaction, leading to reduced ubiquitination and accumulation of GLUT1 at the plasma membrane. The resulting increase in glucose uptake and glycolytic flux promotes hepatocellular carcinoma progression.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/42d7528a9f8c259c297ed5b2.png"},{"id":88750503,"identity":"4af28dc2-97b8-466d-b016-16be469425c2","added_by":"auto","created_at":"2025-08-11 05:55:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5296243,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/8777b57e-0972-4e1f-95d6-b53f8b2c7acd.pdf"},{"id":88748917,"identity":"4db14698-386a-4ae3-95d9-e6829c95620b","added_by":"auto","created_at":"2025-08-11 05:31:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27954873,"visible":true,"origin":"","legend":"supplementary figures","description":"","filename":"20250709supplementalfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/6776cdb9277148dde058b380.docx"},{"id":88748901,"identity":"021b92d0-8a9f-41da-875b-9c461917494a","added_by":"auto","created_at":"2025-08-11 05:31:51","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1052254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-7102103/v1/5c6ef6f307edbc33b7bba01b.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"ALDH2 Suppresses Glycolysis in Hepatocellular Carcinoma via TRIM21-Mediated GLUT1 Degradation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC), the most common type of primary liver cancer, ranks among the leading causes of cancer-related death worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite advances in diagnosis and treatment, its prognosis remains poor, mainly due to late-stage detection and limited therapeutic options\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. A deeper understanding of the molecular mechanisms driving HCC is critical for developing more effective targeted therapies.\u003c/p\u003e\u003cp\u003eAldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that catalyzes the oxidation of acetaldehyde, a byproduct of alcohol metabolism, and detoxifies lipid peroxidation\u0026ndash;derived aldehydes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. A common single nucleotide polymorphism (SNP), rs671, results in a glutamic acid\u0026ndash;to\u0026ndash;lysine substitution at position 487 and is present in approximately 30\u0026ndash;50% of East Asians and 8% of the global population\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This variant markedly reduces ALDH2 enzymatic activity and protein stability, leading to reduced ALDH2 expression\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Clinically, ALDH2 expression is significantly downregulated in HCC and correlates with poor prognosis, particularly in individuals with alcohol exposure\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Paradoxically, rs671 carriers tend to consume less alcohol due to adverse reactions, such as facial flushing\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, suggesting that ALDH2 may also have alcohol-independent role in liver tumorigenesis. Our previous work has demonstrated non-catalytic functions of ALDH2 in lipid metabolism\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. however, whether and how ALDH2 contributes to HCC progression independently of alcohol metabolism remains unknown.\u003c/p\u003e\u003cp\u003eMetabolic reprogramming is a hallmark of cancer, and many tumors rely on elevated aerobic glycolysis (the Warburg effect) to support rapid proliferation\u003csup\u003e16 17\u003c/sup\u003e. Increased glucose uptake is a prerequisite for this phenotype and is often driven by elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. GLUT1 is frequently upregulated in HCC and is associated with poor clinical outcomes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, highlighting its potential as a therapeutic target. However, the upstream mechanisms that regulate GLUT1 stability in HCC remain poorly understood.\u003c/p\u003e\u003cp\u003eHere, we identify a previously unrecognized, alcohol-independent role of ALDH2 in suppressing HCC progression through post-translational regulation of GLUT1. Mechanistically, ALDH2 promotes the recruitment of the E3 ubiquitin ligase TRIM21 to GLUT1, facilitating K48-linked polyubiquitination at lysine 256 (K256) and subsequent proteasomal degradation. Loss of ALDH2\u0026mdash;via genetic deletion or the rs671 variant\u0026mdash;disrupts this regulatory axis, resulting in GLUT1 accumulation, enhanced glycolytic flux, and accelerated tumor growth. Notably, pharmacological inhibition of GLUT1 with BAY-876 significantly suppresses tumor development in both xenograft and DEN-induced HCC mouse models with ALDH2 deficiency, highlighting a potential therapeutic strategy targeting metabolic vulnerabilities in ALDH2-deficient liver cancer.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eALDH2 is downregulated in hepatocellular carcinoma and predicts poor prognosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the clinical relevance of ALDH2 in HCC, we analyzed its transcript levels in publicly available datasets. Both the TCGA-LIHC and GSE14520 cohorts revealed a significant reduction in ALDH2 mRNA expression in HCC tumor tissues compared to adjacent normal liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;B). Kaplan\u0026ndash;Meier survival analysis further demonstrated that low ALDH2 expression was associated with reduced overall survival in HCC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These findings were validated at the protein level by immunoblotting of 24 paired human HCC and adjacent non-tumor liver tissues, which confirmed a marked downregulation of ALDH2 in tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026ndash;E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the functional role of ALDH2 in HCC, we utilized Aldh2 global knockout mice and subjected them to diethylnitrosamine (DEN)-induced liver tumorigenesis. Efficient ALDH2 knockout in liver tissues was confirmed by immunoblotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Compared to wild-type controls, AKO mice exhibited significantly increased tumor burden, as evidenced by elevated liver-to-body weight ratios, increased tumor number, and greater maximum tumor volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG\u0026ndash;J and Supplemental Fig.\u0026nbsp;1F).\u003c/p\u003e\u003cp\u003eTo extend these findings to a clinically relevant genetic context, we utilized Aldh2 rs671 knock-in mice (E504K), which model the common East Asian loss-of-function variant\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Consistent with the knockout model, rs671 mice exhibited reduced hepatic ALDH2 protein expression (Supplementary Fig.\u0026nbsp;1A) and developed more numerous and larger liver tumors following DEN administration, with a shifted tumor number distribution (Supplementary Fig.\u0026nbsp;1B\u0026ndash;E). Together, these results establish that ALDH2 deficiency promotes HCC development in an alcohol-independent manner.\u003c/p\u003e\u003cp\u003e\u003cb\u003eALDH2 loss enhances glucose metabolism in HCC\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the metabolic consequences of ALDH2 deficiency in HCC under non-alcoholic conditions, we performed untargeted metabolomic profiling of liver tissues from WT and AKO mice following DEN treatment, using a high-resolution mass spectrometry-based platform previously established in our laboratory\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Unsupervised clustering of significantly altered metabolites revealed marked changes in carbohydrate and nucleotide metabolism in AKO livers compared to WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Pathway enrichment analysis further indicated upregulation of the Warburg effect and the tricarboxylic acid (TCA) cycle pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting a metabolic shift toward enhanced glucose utilization in ALDH2-deficient HCC livers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further characterize these alterations, we conducted [U-\u0026sup1;\u0026sup3;C₆]-glucose tracing in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Compared with WT controls, both AKO and rs671 knock-in livers showed significantly increased \u0026sup1;\u0026sup3;C enrichment in glycolytic intermediates\u0026mdash;pyruvate, lactate, and alanine\u0026mdash;indicating elevated glycolytic flux (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;E). Additionally, enhanced labeling of TCA cycle\u0026ndash;derived metabolites, such as citrate, proline, fumarate, malate, aspartate, and glutamate, was observed in both models (Supplementary Fig.\u0026nbsp;2A\u0026ndash;B), indicating increased glucose oxidation through mitochondrial metabolism. These findings were further validated in vitro using HCC cells cultured with [U-\u0026sup1;\u0026sup3;C₆]-glucose, ALDH2 knockdown led to elevated incorporation of \u0026sup1;\u0026sup3;C into both secreted glycolytic products and intracellular TCA intermediates, including citrate, malate, and glutamate (Supplementary Fig.\u0026nbsp;2C\u0026ndash;E).\u003c/p\u003e\u003cp\u003eTo assess whether altered glucose uptake\u0026mdash;a critical initiating step of glycolysis\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u0026mdash;underlies this phenotype, we measured 2-NBDG-6-phosphate (2-NBDG) uptake. ALDH2 knockdown significantly enhanced glucose uptake in LPC-H12 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Consistently, ALDH2-deficient cells consumed more glucose from the culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), supporting a state of increased glucose demand driven by ALDH2 loss.\u003c/p\u003e\u003cp\u003e\u003cb\u003eALDH2 loss promotes GLUT1-driven glucose metabolic activation in HCC\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe observed increase in glucose flux prompted us to investigate the underlying mechanisms driving enhanced glucose metabolism in ALDH2-deficient HCC. Given that glucose transporter 1 (GLUT1) is a key rate-limiting factor for glucose uptake in cancer cells, we examined its expression across multiple models. Immunoblotting revealed markedly elevated GLUT1 protein levels in DEN-induced tumors from ALDH2 knockout and rs671 knock-in mice compared to wild-type controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;3A). Similarly, stable ALDH2 knockdown in LPC-H12 and Hepa1-6 HCC cells increased GLUT1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These findings were further supported by human clinical samples, in which tumors with reduced ALDH2 expression showed frequent upregulation of GLUT1 protein compared to matched adjacent non-tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Despite consistent GLUT1 protein upregulation, \u003cem\u003eGlut1\u003c/em\u003e mRNA levels remained unchanged in ALDH2-deficient mouse livers, rs671 mice, and HCC cell lines (Supplementary Fig.\u0026nbsp;3B\u0026ndash;C), suggesting post-transcriptional regulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further examine the impact of the rs671 variant on GLUT1 regulation in human HCC, we stratified 50 paired tumor samples by ALDH2 genotype (WT vs rs671) and performed immunoblotting. Quantification revealed higher GLUT1 protein levels in rs671 tumors compared to WT counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;3D). Analysis of TCGA data corroborated our findings, showing that SLC2A1 (GLUT1) is significantly upregulated in HCC tumors relative to normal liver (Supplementary Fig.\u0026nbsp;3E). Across the patient cohort, ALDH2 and GLUT1 protein levels were inversely correlated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), supporting a conserved regulatory relationship in human HCC.\u003c/p\u003e\u003cp\u003eAs GLUT1-mediated glucose transport requires translocation to the plasma membrane, we next examined whether ALDH2 deficiency alters GLUT1 subcellular distribution. Fractionation experiments revealed enrichment of GLUT1 in the plasma membrane (PM) fraction of ALDH2-deficient samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. F-G and Supplemental Fig.\u0026nbsp;3G), a finding further supported by immunofluorescence in ALDH2-knockdown HCC cells (Supplemental Fig.\u0026nbsp;3F).\u003c/p\u003e\u003cp\u003eTo determine whether GLUT1 is functionally required for the metabolic phenotype induced by ALDH2 loss, we treated cells with BAY-876, a selective GLUT1 inhibitor\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. BAY-876 administration significantly reduced glucose consumption (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), attenuated \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC incorporation from [U-\u0026sup1;\u0026sup3;C₆]-glucose into glycolytic and TCA intermediates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), and suppressed lactate secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ), confirming the dependence of enhanced glucose metabolism on GLUT1 activity.\u003c/p\u003e\u003cp\u003eIn addition, patients with high GLUT1 expression exhibited significantly poorer overall survival (Supplementary Fig.\u0026nbsp;3H). To further evaluate the functional relevance of GLUT1 in hepatoma cells, we performed siRNA-mediated knockdown in parental LPC-H12 cells. GLUT1 silencing markedly suppressed cell proliferation, confirming its role as a driver of HCC cell growth independent of ALDH2 status (Supplementary Fig.\u0026nbsp;3I). Consistently, pharmacological inhibition of GLUT1 using the selective inhibitor BAY-876 resulted in a dose-dependent reduction in cell proliferation (Supplementary Fig.\u0026nbsp;3J), further supporting the therapeutic potential of targeting GLUT1. Moreover, patients with concurrent low ALDH2 and high GLUT1 expression had the poorest prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK), reinforcing the clinical relevance of the ALDH2\u0026ndash;GLUT1 regulatory axis in HCC progression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eALDH2 deficiency stabilizes GLUT1 by reducing GLUT1 ubiquitination\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTo investigate how ALDH2 regulates GLUT1 protein abundance, we first assessed its effect on GLUT1 protein turnover. Cycloheximide (CHX) chase assays showed that GLUT1 degradation was significantly delayed in ALDH2-knockdown cells compared to controls, suggesting that ALDH2 promotes GLUT1 degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;B). To determine the relevant degradation pathway, we treated cells with either the proteasome inhibitor MG132 or the lysosome inhibitor chloroquine (CQ) (Supplementary Fig.\u0026nbsp;4A). MG132, but not CQ, effectively blocked GLUT1 degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;D; Supplementary Fig.\u0026nbsp;4B), suggesting that ALDH2 promotes GLUT1 turnover primarily through the ubiquitin\u0026ndash;proteasome system.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next examined whether ALDH2 modulates GLUT1 ubiquitination. Immunoprecipitation of endogenous GLUT1 revealed reduced GLUT1 ubiquitination in both ALDH2-deficient liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;F) and ALDH2 knockdown LPC-H12 cells (Supplementary Fig.\u0026nbsp;4C), indicating that ALDH2 facilitates GLUT1 ubiquitination in both in vivo and in vitro. To confirm this in a defined system, we co-expressed V5-tagged GLUT1, HA-tagged ubiquitin, and either wild-type or rs671-mutant FLAG-tagged ALDH2 in 293T cells. Both ALDH2 constructs increased GLUT1 ubiquitination to a similar extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), suggesting that ALDH2\u0026rsquo;s enzymatic activity is not required for this function.\u003c/p\u003e\u003cp\u003eGiven prior findings that that ALDH2 can bind to Rac2 and influence its degradation \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, we hypothesized that ALDH2 may similarly interact with GLUT1 to facilitate its proteasomal degradation. Co-immunoprecipitation assays in 293T cells confirmed that both wild-type and rs671 ALDH2 can bind with GLUT1 (Supplementary Fig.\u0026nbsp;4D\u0026ndash;E). This interaction was also detected in wild-type mouse liver tissues and LPC-H12 cells but was markedly reduced in ALDH2-deficient (AKO or rs671) samples, likely due to reduced ALDH2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH\u0026ndash;I; Supplementary Fig.\u0026nbsp;4F). Importantly, endogenous ALDH2\u0026ndash;GLUT1 binding was also observed in paired human HCC tumor and adjacent non-tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e\u003cp\u003eTogether, these findings demonstrate that ALDH2 interacts with GLUT1 and promotes its ubiquitin-dependent proteasomal degradation. Loss of ALDH2\u0026mdash;either via gene knockout or the rs671 variant\u0026mdash;impairs GLUT1 ubiquitination, leading to its stabilization in liver and HCC cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe ALDH2\u0026ndash;TRIM21 complex catalyzes K48-linked ubiquitination of GLUT1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify the E3 ligase responsible for GLUT1 ubiquitination downstream of ALDH2, we performed mass spectrometry-based proteomic analysis following immunoprecipitation of ALDH2- and GLUT1-associated protein complexes. TRIM21 and MUL1 emerged as top candidate E3 ligases that potentially interact with both ALDH2 and GLUT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Supplementary Fig.\u0026nbsp;5A\u0026ndash;B). TRIM21 and MUL1 are conserved E3 ubiquitin-protein ligases known to regulate diverse signaling pathways including OCT1, p53, NF-κB, and HIF-1α\u003csup\u003e28\u003c/sup\u003e. To evaluate their role in GLUT1 regulation, we silenced each ligase in LPC-H12 cells with or without ALDH2 knockdown. GLUT1 levels increased upon the knockdown of either ligase, suggesting both contribute to GLUT1 degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). However, in co-transfected 293T cells, TRIM21 induced stronger GLUT1 ubiquitination than MUL1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), a finding confirmed by endogenous ubiquitination assays (Supplementary Fig.\u0026nbsp;5C), supporting TRIM21 as the dominant E3 ligase in this context.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that RING domain of TRIM21 is essential for its catalytic activity\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, we used a RING-deficient TRIM21 mutant (ΔRING). Overexpression of wild-type TRIM21 increased GLUT1 ubiquitination, which was lost with the ΔRING mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), confirming that TRIM21 E3 ligase activity is required.\u003c/p\u003e\u003cp\u003eTo determine how ALDH2 facilitates this process, we assessed complex formation between ALDH2, TRIM21, and GLUT1. Co-immunoprecipitation in 293T cells showed that TRIM21 interacts with both ALDH2 and GLUT1 (Supplementary Fig.\u0026nbsp;5D\u0026ndash;E), and that this interaction is preserved in the ALDH2 rs671 variant, indicating that enzymatic activity is dispensable. In liver tissues from wild-type, ALDH2-knockout, and rs671 mice, TRIM21-associated GLUT1 and ALDH2 levels were significantly reduced in ALDH2-deficient conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Similar reductions were observed in ALDH2-knockdown LPC-H12 cells (Supplementary Fig.\u0026nbsp;5F). In human HCC specimens, TRIM21 co-immunoprecipitated both ALDH2 and GLUT1 in adjacent non-tumor tissues, while these interactions were diminished in matched tumor samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;G). Reciprocal IP using anti-ALDH2 antibody further confirmed reduced interaction with TRIM21 and GLUT1 in tumors tissues (Supplementary Fig.\u0026nbsp;5G), supporting the idea that ALDH2 serves as a scaffold stabilizing the TRIM21\u0026ndash;GLUT1 interaction.\u003c/p\u003e\u003cp\u003eTo directly test this mechanism, we performed \u003cem\u003ein vitro\u003c/em\u003e ubiquitination assays using purified components. TRIM21 catalyzed polyubiquitination of GLUT1, which was abolished by ΔRING mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Addition of recombinant ALDH2 further enhanced GLUT1 ubiquitination in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), indicating that ALDH2 promotes TRIM21 activity.\u003c/p\u003e\u003cp\u003eNext, we assessed linkage specificity. K48-linked polyubiquitin chains are canonical signals for proteasomal degradation \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. TRIM21 selectively induced K48-linked\u0026mdash;but not K63-linked\u0026mdash;ubiquitination of GLUT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ) and did not promote ubiquitination through other lysine linkages (K6, K11, K27, K29, K33; Supplementary Fig.\u0026nbsp;5H). Finally, both wild-type and rs671 ALDH2 enhanced TRIM21-mediated K48 ubiquitination to a similar degree (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK), demonstrating that this function is independent of ALDH2 enzymatic activity.\u003c/p\u003e\u003cp\u003eTogether, these results identify TRIM21 as the primary E3 ligase responsible for GLUT1 ubiquitination and reveal that ALDH2, through protein\u0026ndash;protein interaction, facilitates TRIM21-mediated K48-linked ubiquitination of GLUT1, thereby promoting its degradation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eUbiquitination of GLUT1 at K256 is required for ALDH2-induced degradation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify the specific ubiquitination site responsible for GLUT1 degradation, we performed mass spectrometry analysis and identified lysine 256 (K256) as a candidate ubiquitination site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Sequence alignment showed that K256 is highly conserved across multiple species (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), suggesting its potential functional importance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate its role in GLUT1 stability, we conducted cycloheximide (CHX) chase assays in cells expressing either wild-type or K256R-mutant GLUT1. The K256R mutant exhibited markedly increased stability compared to WT, indicating that K256 is a critical for GLUT1 degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eWe next tested whether K256 is required for TRIM21-mediated ubiquitination. In 293T cells co-transfected with HA-tagged TRIM21 and V5-tagged GLUT1, the K256R mutation significantly impaired TRIM21-induced GLUT1 ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). This was further validated using an in vitro reconstituted ubiquitination system, where recombinant TRIM21 and ALDH2 efficiently catalyzed polyubiquitination of WT-GLUT1, but not the K256R mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Together, these findings identify K256 as the principal site for TRIM21-dependent ubiquitination of GLUT1.\u003c/p\u003e\u003cp\u003eTo assess the functional relevance of K256 ubiquitination in vivo, we generated subcutaneous xenograft models using Hep1-6 cells stably expressing either GLUT1^WT or GLUT1^K256R, with or without ALDH2 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Immunoblotting confirmed successful GLUT1 expression overexpression in tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). ALDH2 overexpression significantly reduced tumor growth In GLUT1^WT tumors, but this effect was abolished in GLUT1^K256R tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH\u0026ndash;J), supporting a model in which ALDH2 suppresses tumorigenesis via K256-dependent ubiquitination and degradation of GLUT1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGLUT1 inhibition reverses tumor-promoting effects of ALDH2 deficiency\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the therapeutic relevance of GLUT1 in ALDH2-deficient HCC, we first used a subcutaneous xenograft model with Hepa1-6 cells stably expressing shALDH2. Mice were treated with the selective GLUT1 inhibitor BAY-876 (4 mg/kg, oral gavage once daily), starting on day 7 post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). ALDH2 knockdown significantly accelerated tumor growth, while BAY-876 treatment significantly suppressed tumor progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u0026ndash;D), suggesting that ALDH2 deficiency drives tumorigenesis through a GLUT1-dependent mechanism amenable to pharmacological inhibition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further confirm this dependency, we genetated a dual-knockdown model targeting both ALDH2 and GLUT1 in Hepa1-6 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026ndash;F). Genetic silencing of GLUT1 in the ALDH2-deficient cells significantly reduced tumor volume and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG\u0026ndash;I), consistent with the effect of BAY-876 treatment. These findings collectively support GLUT1 as a critical effector of ALDH2 loss in promoting tumor growth.\u003c/p\u003e\u003cp\u003eTo validate these results in a more physiologically relevant setting, we treated DEN-induced HCC mice (AKO and rs671) with BAY-876 beginning at week 28 (Supplementary Fig.\u0026nbsp;6A, F). No significant weight differences were observed post-treatment (Supplemental Fig.\u0026nbsp;7, A and D), indicating that the dosage was safe. BAY-876 significantly reduced tumor number and burden in both ALDH2 knockout and rs671 knock-in mice, while exerting minimal effects in wild-type controls (Supplementary Fig.\u0026nbsp;6B\u0026ndash;D, G\u0026ndash;I and Supplementary Fig.\u0026nbsp;7C, F). This selective response is likely due to lower baseline GLUT1 expression in wild-type livers, which limits the efficacy of GLUT1 inhibition in the presence of functional ALDH2.\u003c/p\u003e\u003cp\u003eStable isotope tracing further demonstrated that BAY-876 treatment attenuated the elevated glycolytic and TCA fluxes observed in ALDH2-deficient livers (Supplementary Fig.\u0026nbsp;6E, J; Supplementary Fig.\u0026nbsp;7B, E), confirming the mechanistic link between GLUT1 upregulation and metabolic reprogramming upon ALDH2 loss.\u003c/p\u003e\u003cp\u003eTogether, these data demonstrate that ALDH2 deficiency promotes tumorigenesis by enhancing GLUT1 expression and glucose metabolism, and that targeting GLUT1\u0026mdash;genetically or pharmacologically\u0026mdash;effectively suppresses tumor growth in ALDH2-deficient HCC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough surgical and systemic therapies have improved survival in hepatocellular carcinoma (HCC), overall outcomes remain suboptimal compared to other common cancers \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. A better understanding of tumorigenic mechanisms and the identification of precision biomarkers are critical to advance early diagnosis and precision therapies. Metabolic reprogramming, particularly the upregulation of glycolysis, is a hallmark of HCC that supports the energetic and biosynthetic demands. The ALDH2 rs671 variant is associated with poor HCC prognosis, especially in East Asian populations\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. While most studies have focused on the accumulation of acetaldehydes due to reduced enzymatic activity of ALDH2 rs671, emerging evidence suggests that ALDH2 may also exert non-enzymatic functions independent of alcohol metabolism. In this study, we uncover previously unrecognized, non-catalytic function of ALDH2 in modulating glucose metabolism through regulation of GLUT1 stability. Mechanistically, ALDH2 facilitates the recruitment of the E3 ligase TRIM21, which catalyzes K48-linked polyubiquitination of GLUT1 at lysine 256, leading to its proteasomal degradation. ALDH2 deficiency\u0026mdash;either by knockout or the rs671 variant\u0026mdash;disrupts this interaction, resulting in GLUT1 stabilization and enhanced glycolytic activity in HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings reveal a tumor-suppressive role of ALDH2 beyond its canonical mitochondrial function.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough metabolic reprogramming is well established in cancer biology, the upstream regulatory mechanisms governing glucose metabolism in HCC remain poorly defined\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our prior work demonstrated that ALDH2 regulates lipid metabolism by stabilizing HMGCR, the rate-limiting enzyme in cholesterol biosynthesis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. While earlier studies linked ALDH2 to glucose metabolism in the context of alcohol exposure\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, its role in non-alcoholic HCC has not been explored. Here, using metabolomics and [U-\u0026sup1;\u0026sup3;C₆]-glucose tracing, we show that ALDH2 deficiency enhances glycolytic and TCA cycle fluxes via GLUT1 stabilization, supporting a Warburg-like metabolic phenotype.\u003c/p\u003e\u003cp\u003eOur findings also show translational significance. GLUT1 is frequently overexpressed in HCC and correlates with poor clinical outcomes\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. We demonstrate that ALDH2 deficiency increases GLUT1 protein levels and that genetic or pharmacological inhibition of GLUT1 suppresses tumor growth in ALDH2-deficient models. These results position ALDH2 and GLUT1 expression levels as potential prognostic markers and highlight GLUT1 as a therapeutic vulnerability, particularly in patients harboring the rs671 variant.\u003c/p\u003e\u003cp\u003ePost-translational modifications are known to control GLUT1 trafficking and degradation. For instance, phosphorylation at Ser226 by protein kinase C promotes membrane localization\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Ubiquitination represents another regulatory mechanism. TRIM21, a RING-type E3 ligase, has been implicated in the turnover of metabolic enzymes including FASN, PFK1, and G6PD \u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45 CR46\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. While PP2Aα has been reported to inhibit TRIM21-mediated GLUT1 degradation through dephosphorylation\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, the specific linkage type and ubiquitination site were previously unknown. Our study identifies K48-linked polyubiquitination at lysine 256 as the dominant modification and implicates ALDH2 as a molecular scaffold facilitating this process. Interestingly, ALDH2 is not confined to mitochondria\u0026mdash;it may also localize to the cytosol and nucleus\u0026mdash;suggesting broader regulatory potential across subcellular compartments.\u003c/p\u003e\u003cp\u003eIn summary, we define a novel tumor-suppressive function of ALDH2 in repressing glucose metabolism and tumor progression in HCC. ALDH2 deficiency, as modeled by the rs671 variant, disrupts TRIM21-mediated ubiquitination of GLUT1, promoting metabolic activation and tumorigenesis. Targeting the ALDH2\u0026ndash;TRIM21\u0026ndash;GLUT1 axis may offer a promising therapeutic avenue, particularly for genetically predisposed patient populations.\u003c/p\u003e\u003cp\u003eSeveral questions remain. The structural basis of ALDH2\u0026ndash;TRIM21\u0026ndash;GLUT1 interactions requires further elucidation. It is also unclear whether this regulatory mechanism is conserved in other GLUT1-high tumors. Finally, although BAY-876 showed therapeutic efficacy \u003cem\u003ein vivo\u003c/em\u003e, its pharmacokinetics, toxicity profile, and potential synergy with existing therapies warrant further investigation. Future studies should also explore the therapeutic feasibility of restoring ALDH2 function in metabolically reprogrammed tumors.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All animal procedures were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences. Mice were housed in a temperature-controlled and pathogen-free environment with a 12-hour light/12-hour dark cycle. ALDH2-KO mice were a kind gift from Drs. Jun Ren and Aijun Sun (Zhongshan Hospital, Fudan University, Shanghai, China)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Rs671-KI mice were a gift from Dr. Yong Cang (ShanghaiTech University, Shanghai, China)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Genome-edited F0 ALDH2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were backcrossed to C57BL/6J mice for two generations. Wild-type and ALDH2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were derived from crossing ALDH2\u003csup\u003e+/\u0026minus;\u003c/sup\u003e mice. WT and ALDH2*2 (rs671, homozygous) mice were obtained by breeding ALDH2*1/*2 mice. AKO, ALDH2 rs671, and their respective control mice were fed a standard diet (P1101F-25).\u003c/p\u003e\u003cp\u003eHepatocellular carcinoma (HCC) mouse models were generated by intraperitoneal injection of 50 \u0026micro;g/g body weight of diethylnitrosamine (DEN) on postnatal day 14. Mice were euthanized at 37 weeks of age, and the liver tumor burden was assessed by quantifying visible tumor volume and number.\u003c/p\u003e\u003cp\u003e\u003cb\u003eXenograft mouse model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFour-week-old male athymic nude mice were purchased from Shanghai Slack Laboratory Animal Corp. Ltd. Hepa1-6 cells (2 \u0026times; 10⁶) were resuspended in a 1:1 mixture of DMEM and Matrigel (Corning, 356234) and injected subcutaneously into the mice. Tumor dimensions were assessed every other day, and tumor volume was calculated using the formula: volume (mm\u0026sup3;)\u0026thinsp;=\u0026thinsp;0.52 \u0026times; length \u0026times; width\u0026sup2;.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMice tracing experiment with\u003c/b\u003e \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003e6\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-glucose\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMice were fasted for 12 hours prior to euthanasia. They were injected intravenously with 1 g/kg [U-13C₆]-glucose (CLM-1396-PK, Cambridge Isotope Laboratories) via the tail vein at 15-minute intervals over a 30-minute labeling period. Liver tissues were promptly harvested for metabolic profiling.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical inhibition of GLUT1 in mice models\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWT and AKO mice were injected with 50 \u0026micro;g/g DEN on postnatal day 14. At week 28, mice were stratified into two cohorts. One group was orally administered BAY-876 (Selleck, S8452) at 5 mg/kg daily from week 28 to week 32, while the control group received vehicle (0.5% CMC-Na). Tumor volume was monitored throughout the treatment period. Plasma and liver tissues were collected for further experimental analysis.\u003c/p\u003e\u003cp\u003eFor the xenograft model, mice were randomized into two groups when tumor volumes reached approximately 100 mm\u0026sup3;. Mice were orally administered either vehicle or 4 mg/kg BAY-876 daily until the study endpoint. BAY-876 was dissolved in 0.5% CMC-Na (Selleck, S6703), which was prepared in sterile saline.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culture, transfection and drug treatments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell lines were obtained from the CAS Cell Bank and cultured in DMEM (Gibco, C11995500BT) supplemented with 10% FBS (Gibco, 10099141) and 1% penicillin\u0026ndash;streptomycin (Invitrogen, 15140-122) in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e\u003cp\u003eTransfection was performed using Lipofectamine 3000 (Thermofisher, L3000015) and Liji sheng wu (AC04L011) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003eFor drug treatments, MG132 (S2619) and CQ (S6999) were purchased from Selleck. CHX (HY-12320) was purchased from MCE. Medium containing a final concentration were then used to replace the original medium and cells were cultured in the presence of drugs for the indicated times were described in detail in the figure legend.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolic flux experiments using [U-\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC\u003c/b\u003e\u003csub\u003e\u003cb\u003e6\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e]-glucose and gas chromatography/ mass spectrometry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe metabolic flux analysis using [U-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC\u003csub\u003e6]\u003c/sub\u003e-glucose followed our previously published protocol\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. For stable isotope labeling, cells were seeded at a density of approximately 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per 10-cm dish. When cells reached\u0026thinsp;~\u0026thinsp;70% confluence, the unlabeled medium was replaced with labeling medium for indicated time. The labeling medium consisted of low glucose DMEM (Gibco,11054\u0026ndash;020: 1 g/L unlabeled glucose, no glutamine) with supplement of 1 g/L [U-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e]-glucose (CLM-1396-PK, Cambridge Isotope Laboratory), 10% (v/v) fetal bovine serum, 1 mM pyruvate, 2 mM unlabeled L-glutamine, and 1% (v/v) penicillin-streptomycin.\u003c/p\u003e\u003cp\u003eFor sample pretreatment, cell pellets and 100 \u0026micro;L of medium, were resuspended in 400 \u0026micro;l of pre-colded 50% methanol/50% water containing 100 \u0026micro;M Norvaline (internal standard). The mixture was then thoroughly mixed, and rapidly frozen in dry ice for 30 min. Subsequently, 300 \u0026micro;l of chloroform was added, vortexed for 30 s, and centrifuged at 12,000 rpm at 4\u0026deg;C for 10 min. The upper aqueous phase was collected, freeze-concentrated, and dried.\u003c/p\u003e\u003cp\u003eFor tissue samples pretreatment, 30 \u0026micro;g of tissue was added to 400 \u0026micro;l of pre-colded 50% methanol/50% water containing 100 \u0026micro;M Norvaline. The sample was then homogenized using magnetic beads, immediately placed on dry ice for 30 minutes, and subsequently processed in the same manner as the cell samples.\u003c/p\u003e\u003cp\u003eFor sample derivatization, 20 mg/ml O-Isobutylhydroxylamine Hydrochloride (sigma, CIAH987ED435) was prepared in pyridine to protect ketone moieties. Each sample tube was supplemented with 70 \u0026micro;l of keto-protecting reagent, was thoroughly mixed, and incubated in a metal bath at 85\u0026deg;C for 20 minutes. After cooling, 30 \u0026micro;l of the derivating agent BSMFT (N-tert-Butyldimethylsilyl-N-Methytrifluoro-acetamide, sigma, 394882) was added to each tube, vortexed and incubated in a metal bath at 85\u0026deg;C for 60 min. Centrifuge at 12,000 rpm for 10 minutes at 4\u0026deg;C, transfer the supernatant to a vial for GC/MS analysis.\u003c/p\u003e\u003cp\u003eGC-MS analysis was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. A Shimadzu QP-2010 Ultra GC-MS was programmed with an injection temperature of 250\u0026deg;C injection split ratio 1/10 and injected with 1 \u0026micro;L of sample. The GC column used was a 30 m \u0026times; 0.25 mm \u0026times; 0.25 mm HP-5ms. GC-MS data were analysed to determine isotope labelling andquantities of metabolites and implemented in MATLAB\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were incubated in cell lysis (25 mM Tris-HCL, 150 mM NaCL, containing protease inhibitor cocktail and 2% (w/v) n-dodecyl-b-D-maltoside (APExBIO, C4421) for 2 h at 4 \u003csup\u003e◦\u003c/sup\u003eC and tissues were homogenized in 500 \u0026micro;L tissue lysis buffer. The soluble fractions of cell lysates were isolated by centrifugation at 4\u0026deg;C, 12,000 g for 10 min. Protein concentrations were determined using a protein assay kit (Beyotime, P0010). The supernatants were boiled for 10 min at 37\u0026deg;C. Proteins were separated by SDS-PAGE and transferred onto 0.45 \u0026micro;M PVDF membranes (Millipore, IPFL00005). After extensive rinse with TBST, immunoreactive bands were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using a chemiluminescence (ECL) kit (Proteintech) through Tanon 5200 Chemiluminescent Imaging System. All western blots were analyzed with ImageJ for quantitative measurements.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibodies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe following antibodies were used for immunoblotting. Anti-ALDH2 (15310-1-AP, 1:1000), Anti-GAPDH (60004-1-Ig, 1:5000), Anti-HA-Tag (66006-2-Ig, 1:1000), Anti-VINCULIN (26520-1-AP, 1:1000), Anti-FLAG-Tag (20543-1-AP, 1:1000) were purchased from Proteintech. Anti-GLUT1 (12939S, 1:1000), and normal rabbit IgG (2729S) were purchased from Cell Signaling Technology. Anti-GLUT1 (ab115730, 1:1000), were purchased from Abcam. Anti-Ubiquitin (Sc-8017, 1:500), and V5-Tag (Sc-81594, 1:1000) were purchased from Santa Cruz.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunoprecipitation experiments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHEK293T cells were transfected with target tagged protein using Attracting Transfection reagent (AC04L011, Liji shengwu), incubated by IP cell lysis buffer (50 mM Tris-HCL, 400 mM NaCl, 0.8% Triton X-100, pH 7.5), and cleared with targeted beads overnight. For LPC-H12 hepatocytes and liver tissues, cell lysates were incubated with target primary antibodies overnight at 4℃. Then protein A/G beads (B23202, Bimake) were added to each group and incubated with protein suspension for 4h at 4℃. Beads were washed with a wash buffer (50 mM Tris-HCl, 400Mm NaCl and 0.8% Triton-X-100, pH 7.5) three to five times. Proteins were eluted in sample buffer at 95℃ for 10 min and separated by SDS-PAGE and transferred to PVDF membrane for immunoblotting.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of interacting proteins with GLUT1 by using iTRAQ proteomics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter immunoprecipitation for GLUT1 in LPC-H12 cells, we separated total cell lysate by using 10% SDS-PAGE for silver stain (catalog P0017S, Beyotime). The gel was extracted, and the proteins were identified using iTRAQ proteomics by Novognen.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescence assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOn the day prior to the experiment, the clean coverslips were immersed in 75% ethanol and then air dried and placed at the base of a six-well plate on the ultra-clean bench under a UV lamp for 6\u0026ndash;12 hours to ensure sterility. On the day of the experiment, cells were seeded into the sterilized six-well plate with a cell density of 30%-50% with uniform distribution. Once the cells adhere to the surface, the culture medium was discarded and gently rinsed once with pre-chilled 1\u0026times;PBS. The cells were fixed with 1 mL pre-chilled 4% PFA at room temperature for 10\u0026ndash;20 min. After discarding the fixative, 1 mL of 1\u0026times;PBS were gently added and placed on a shaker for 5 min. This process was repeated three times. One mL of 1% Triton X-100 was added at room temperature and incubated for 10 min. After discarding the permeabilization solution, 1 mL pre-chilled 1\u0026times;PBS were washed on a shaker for 5 min. After three washes, 1 mL of 1% BSA was added at room temperature and incubated for 30 min. After discarding the blocking solution, the corresponding primary antibody (diluted according to the instructions) was added and incubated at 4\u0026deg;C overnight. The next day, 1 mL pre-chilled 1\u0026times;PBS was added on a shaker for 5 min. The corresponding secondary antibody was added and incubated at 37\u0026deg;C in the dark for 30 min. After washing with 1 mL pre-chilled 1\u0026times;PBS on a shaker for 5 min and repeating this process three times, DAPI was added for nuclear staining and sealed with transparent nail polish. After drying, images were taken using a Zeiss LSM 780 or stored in the dark at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlasma membrane and intracellular membrane fractionation.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePlasma membrane (PM) and intracellular membrane (ICM) proteins were isolated using the PM/ICM Protein Extraction Kit (ab65400, Abcam) following the manufacturer\u0026rsquo;s protocol. Briefly, cells were washed twice with ice-cold PBS, then incubated with 2 ml of homogenization buffer on ice and collected using a cell scraper. The cell suspension was homogenized on ice using a Dounce homogenizer (30\u0026ndash;50 strokes), followed by centrifugation at 700\u0026times;g for 5 min at 4\u0026deg;C. The supernatant was transferred to fresh tubes and centrifuged at 10,000\u0026times;g for 30 min at 4\u0026deg;C. The resulting pellet contained total membrane proteins, while the supernatant contained cytosolic proteins.\u003c/p\u003e\u003cp\u003eThe membrane pellet was resuspended in 200 \u0026micro;l of upper-phase solution and mixed with 200 \u0026micro;l of lower-phase solution. After incubation on ice for 5 min, samples were centrifuged at 1,000\u0026times;g for 5 min at 4\u0026deg;C to separate the phases. The upper-phase (containing PM proteins) and lower-phase (containing ICM proteins) were collected into separate tubes. To increase yield, phase separation was repeated by adding fresh upper-phase solution to the lower-phase and vice versa. The corresponding phases from both rounds were pooled and diluted with 5 volumes of water, then centrifuged at 15,000\u0026times;g for 30 min at 4\u0026deg;C. The resulting pellets containing enriched PM or ICM proteins were collected for immunoblot analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGlucose uptake by 2-NBDG\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeed cells 24 h prior to the experiment. When the cells reach 70\u0026ndash;80% confluence, starve the cells in HBSS (Gibco, 14025134) medium for 2 h. Aspirate the medium and incubate the cells with 2-NBDG (APExBIO, B6035) solution for 5 min (37\u0026deg;C, 5% CO₂). Terminate the reaction by adding 1 mL of pre-chilled PBS. After obtaining the cell pellet, resuspend the cells in pre-chilled PBS and wash twice to remove unbound 2-NBDG. Finally, resuspend the cells in 500 \u0026micro;L PBS for flow cytometry analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGlucose consumption and lactate production\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe D-Glucose Kit (#10716251035, R-Biopharm) and the L-Lactic Acid Kit (#10139084035, R-Biopharm) were used to detect the concentration of glucose and lactate in the culture medium according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003eIn brief, collect the cell culture media from 0h and 6h time points and place them on ice. For the assay, aliquot 4 \u0026micro;L of each sample, standards, and blanks into a 96-well plate. Add 50 \u0026micro;L of solution 1 and 96 \u0026micro;L of double-distilled water to each well, incubate at room temperature for 10 minutes, and measure the absorbance at 340 nm using a microplate reader; record these values as A1. Subsequently, add 1 \u0026micro;L of suspension 2 to each well and incubate at 37\u0026deg;C for 30 minutes. Measure the absorbance at 340 nm again and record these values as A2. Finally, calculate the glucose concentration in the culture media based on the standard curve.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHuman clinical samples and ALDH2 rs671 genotyping\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTumor tissues and matched adjacent non-tumor tissues from 50 hepatocellular carcinoma (HCC) patients were obtained from the Eastern Hepatobiliary Surgery Hospital (Shanghai, China) and cryopreserved. Frozen tumor tissues were primarily used for protein analysis by western blotting.\u003c/p\u003e\u003cp\u003eAs for genotyping, genomic DNA was extracted from frozen human tissue samples using a commercial tissue DNA extraction kit according to the manufacturer\u0026rsquo;s protocol. DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The extracted genomic DNA was submitted to Bioshine Biotechnology (Hangzhou, China) for Sanger sequencing to determine the genotype of the ALDH2 rs671 polymorphism (c.1510G\u0026thinsp;\u0026gt;\u0026thinsp;A, Glu504Lys). Sequencing chromatograms were analyzed using SnapGene Viewer, and genotypes were classified as wild type (G/G), heterozygous (G/A), or homozygous mutant (A/A) based on the nucleotide at position c.1510 of the ALDH2 reference sequence (NM_000690.2).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell proliferation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, C0005, TargetMol) according to the manufacturer\u0026rsquo;s instructions. For pharmacological inhibition studies, cells were treated with DMSO or BAY-876 (5, 10, 20, or 40 nM; Selleck, S8452) for up to 96 hours. For gene silencing experiments, cells were transfected with siRNA targeting Slc2a1 (si-Slc2a1-1/2/3/4) or negative control siRNA (si-NC) using Lipofectamine 3000 for 24 hours prior to seeding.\u003c/p\u003e\u003cp\u003eCells were seeded at a density of 2 \u0026times; 10\u0026sup3; cells per well in 96-well plates and allowed to adhere for 12 hours. Baseline absorbance (0 h) was measured following incubation with CCK-8 for 1 hour at 37\u0026deg;C, and absorbance at 450 nm was recorded using a microplate reader (BioTek). Cell viability was assessed every 24 hours by replacing the medium with fresh drug-containing medium supplemented with CCK-8. After 1 hour of incubation, absorbance at 450 nm was measured. Absorbance values were normalized to 0 h and background-subtracted using blank wells containing medium and CCK-8 without cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein purification of GST-ALDH2/ GST-ALDH2 rs671, HA-TRIM21/HA-TRIM21ΔR and V5-GLUT1/ V5-GLUT1 K256R\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor bacterial expression, human ALDH2 and ALDH2 RS671 cDNAs were cloned into the pGEX vector to generate N-terminal GST-tagged fusion proteins. The constructs were transformed into E. coli BL21 (DE3) pLysS cells, cultured to an OD₆₀₀ of 0.8, and induced with 0.5 mM IPTG for 7 h at room temperature. Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 200 mM NaCl, 100 mM KCl, 20% glycerol, and 1 mM PMSF) after lysozyme treatment. Lysates were clarified by centrifugation, and recombinant proteins were purified by batch binding to GSTPur Glutathione beads. GST-tagged proteins were eluted with 15 mM reduced glutathione followed by overnight dialysis at 4\u0026deg;C to remove GSH.\u003c/p\u003e\u003cp\u003eFor mammalian expression, HEK293T cells were transfected at ~\u0026thinsp;60% confluency with plasmids encoding HA-tagged TRIM21 or TRIM21ΔR, and V5-tagged GLUT1 or GLUT1 K256R, using Lipofectamine 3000. After 48 h, cells were lysed and recombinant proteins were purified using HA-tag or V5-tag affinity beads (P2185S, P2187S; Beyotime), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro ubiquitination assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUbiquitination reactions were performed in a 60 \u0026micro;L system containing 100 ng E1 (UBE1, 20433ES25, Yeasen), 500 ng E2 (UbcH5b/UBE2D2, HY-P79449, MCE), 500 ng purified E3 ligase (HA-TRIM21 or HA-TRIM21ΔR), 2 \u0026micro;g ubiquitin (20431ES08, Yeasen), 1 \u0026micro;g recombinant substrate protein (V5-GLUT1-His), and 1 \u0026micro;g GST or GST-ALDH2 as indicated. All reactions were carried out in ubiquitination buffer (Mg/ATP buffer, Yeasen) supplemented with 5 mM MgCl₂ and 2 mM ATP. After adding all components, the reaction mixtures were incubated at 30\u0026deg;C for 2 h. Reactions were terminated by adding 1\u0026times; SDS sample loading buffer and incubating at 37\u0026deg;C for 10 min. Samples were then resolved by SDS\u0026ndash;PAGE and analyzed by immunoblotting using anti-Ubiquitin antibodies to detect ubiquitinated forms of GLUT1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eUntargeted Metabolomics Analysis Using UHPLC\u0026ndash;QTOF-MS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUntargeted metabolomics was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Briefly, approximately 20 mg of liver tissue was collected and homogenized in 200 \u0026micro;L of HPLC-grade water using a pre-cooled tissue grinder (60 Hz, 2 minutes). A 10 \u0026micro;L aliquot of the homogenate was reserved for protein concentration measurement for downstream normalization. Quality control (QC) samples were prepared by pooling 10 \u0026micro;L aliquots from each individual homogenate. After homogenization, 800 \u0026micro;L of methanol/acetonitrile (1:1, v/v) containing internal standards was added to each sample, followed by vigorous vortexing for 30 seconds. The mixture was then subjected to ultrasonic homogenization on ice for 10 minutes, followed by snap-freezing in liquid nitrogen for 1 minute. This freeze\u0026ndash;thaw cycle was repeated three times to ensure thorough metabolite extraction. Samples were subsequently stored at \u0026minus;\u0026thinsp;80\u0026deg;C for 1 hour to facilitate protein precipitation. Following centrifugation at 14,000 \u0026times; g for 15 minutes at 4\u0026deg;C, the supernatant was transferred to fresh 1.5 mL tubes, and the solvent was evaporated to dryness under a gentle stream of nitrogen using a nitrogen evaporator. The dried residues were reconstituted in 100 \u0026micro;L of acetonitrile/water (1:1, v/v), centrifuged again at 13,000 \u0026times; g for 15 minutes at 4\u0026deg;C, and 80 \u0026micro;L of the supernatant was transferred into LC\u0026ndash;MS vials for analysis.\u003c/p\u003e\u003cp\u003eMetabolomic profiling was carried out using an ultra-high-performance liquid chromatography (UHPLC) system coupled with a quadrupole time-of-flight (QTOF) mass spectrometer (TripleTOF 6600, SCIEX) operated in both positive and negative ion modes. Chromatographic separation was achieved using a Waters ACQUITY UPLC BEH Amide column (1.7 \u0026micro;m, 100 mm \u0026times; 2.1 mm). The mobile phase composition, gradient program, and flow rate were applied as previously reported. All samples were injected in a random order. Blank samples (100% acetonitrile) and pooled QC samples were run every eight injections to monitor LC\u0026ndash;MS system stability throughout the acquisition.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEthical\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All procedures performed in this study were approved by the Animal Ethics Committee of the SINH, CAS, Shanghai, China. and the Ethics Committee of the Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, China.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eData is expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis was conducted using the unpaired two-tailed Student\u0026rsquo;s t test for two groups by using Graph Pad Prism 9.0. And one-way ANOVA analysis for more than 2 groups. Differences were considered significant at a p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy approval\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, Shanghai, China. All human samples were collected by Department of Hepatic Surgery I (Ward l), Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, China.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eALDH2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ealdehyde dehydrogenase 2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehepatocellular carcinoma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAKO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eALDH2 knockout\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGLUT1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlucose Transporter 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSNP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esingle nucleotide polymorphism\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDEN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ediethyl nitrosamine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e2-NBDG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e2-NBDG-6-phosphate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ewild-type\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTCA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etricarboxylic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCHX\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecycloheximide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCQ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003echloroquine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eepiWAT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eepididymal white adipose tissue\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003esubWAT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esubcutaneous white adipose tissue.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by grants from Shenzhen Medical Research Fund (SMRF B2302042), and National Natural Science Foundation of China (32030053), and RGC General Research Fund (9043653), Startup funds from the City University of Hong Kong (9380154), TBSC Project fund and Futian research project (9609327) and General Basic Research Program of Naval Military Medical University (No. 2023MS039). We are grateful to Prof. Dong Xie and Prof. Jingjing Li (SINH, China) for kindly providing the HA -TRIM21-WT and HA -TRIM21-\u0026Delta;RING expression plasmids.\u0026nbsp;We acknowledge the help from molecular biology core laboratory, animal facilities, and mass spectrometry facilities at Shanghai Institutes of Nutrition and Health (SINH), CAS, Shanghai.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.Y.Y., S.S.Z., and L.L.Z designed this study. L.L.Z. and Y.Q.W performed most of the experiments and data analysis. N.N.L., R.L. performed liquid mass spectrometry analyses. Y.Z.T performed gas mass spectrometry analysis. C.Z.Y. and N.L. provided clinical HCC sample and analysis. X.C., X.Y., X.D.X., L.X.L., Q.C.T., J.W.L., H.M.J., H.R.M., C.X.D., S.T.C. helped with animal study and data analysis. L.L.Z.,\u0026nbsp;H.Y.Y. and S.S.Z drafted and reviewed this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials \u0026amp; Correspondence.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuiyong Yin, Ph.D. Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, China 200031; Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China. Phone: (852) 3442-2812. E-mail: [email protected]\u003c/p\u003e\n\u003cp\u003eShanshan Zhong, Ph.D. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China. E-mail: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVogel A, Meyer T, Sapisochin G, Salem R, Saborowski A (2022) Hepatocellular carcinoma. Lancet 400:1345\u0026ndash;1362\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A et al (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71:209\u0026ndash;249\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGross ER, Zambelli VO, Small BA, Ferreira JC, Chen CH, Mochly-Rosen D (2015) A personalized medicine approach for Asian Americans with the aldehyde dehydrogenase 2*2 variant. Annu Rev Pharmacol Toxicol 55:107\u0026ndash;127\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen CH, Ferreira JC, Gross ER, Mochly-Rosen D (2014) Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev 94:1\u0026ndash;34\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrooks PJ, Enoch M-A, Goldman D, Li T-K, Yokoyama A (2009) The Alcohol Flushing Response: An Unrecognized Risk Factor for Esophageal Cancer from Alcohol Consumption. PLoS Med ;6\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatsuo K, Hamajima N, Shinoda M, Hatooka S, Inoue M, Takezaki T et al (2001) Gene-environment interaction between an aldehyde dehydrogenase-2 (ALDH2) polymorphism and alcohol consumption for the risk of esophageal cancer \u003cem\u003eCarcinogenesis\u003c/em\u003e ;22:913\u0026ndash;916\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFarres J, Wang X, Takahashi K, Cunningham SJ, Wang TT, Weiner H (1994) Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase. J Biol Chem 269:13854\u0026ndash;13860\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJin S, Chen J, Chen L, Histen G, Lin Z, Gross S et al (2015) ALDH2(E487K) mutation increases protein turnover and promotes murine hepatocarcinogenesis. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e ;112:9088\u0026ndash;9093\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao Y, Zhou Z, Ren T, Kim SJ, He Y, Seo W et al (2019) Alcohol inhibits T-cell glucose metabolism and hepatitis in ALDH2-deficient mice and humans: roles of acetaldehyde and glucocorticoids. Gut 68:1311\u0026ndash;1322\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJarl J, Gerdtham UG (2012) Time pattern of reduction in risk of oesophageal cancer following alcohol cessation\u0026ndash;a meta-analysis. Addiction 107:1234\u0026ndash;1243\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTomoda T, Nouso K, Sakai A, Ouchida M, Kobayashi S, Miyahara K et al (2012) Genetic risk of hepatocellular carcinoma in patients with hepatitis C virus: A case control study. J Gastroenterol Hepatol 27:797\u0026ndash;804\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSusumu Higuchi SM, Taro, Muramatsu (1996) Masanobu Murayama, and Motoi Hayashida. Alcohol and Aldehyde Dehydrogenase Genotypes and Drinking Behavior in Japanese. Alcohol Clin Experimental Res\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePautassi RM, Camarini R, Quadros IM, Miczek KA, Israel Y (2010) Genetic and Environmental Influences on Ethanol Consumption: Perspectives From Preclinical Research. Alcoholism: Clin Experimental Res 34:976\u0026ndash;987\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhong S, Li L, Liang N, Zhang L, Xu X, Chen S et al (2021) Acetaldehyde Dehydrogenase 2 regulates HMG-CoA reductase stability and cholesterol synthesis in the liver. Redox Biol 41:101919\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi L, Zhong S, Li R, Liang N, Zhang L, Xia S et al (2022) Aldehyde dehydrogenase 2 and PARP1 interaction modulates hepatic HDL biogenesis by LXRalpha-mediated ABCA1 expression. JCI Insight ;7\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHanahan D (2022) Hallmarks of Cancer: New Dimensions. Cancer Discov 12:31\u0026ndash;46\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324:1029\u0026ndash;1033\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzernin J, Phelps ME (2002) Positron emission tomography scanning: current and future applications. Annu Rev Med 53:89\u0026ndash;112\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoost H-G, Bell GI, Best JD, Birnbaum MJ, Charron MJ, Chen YT et al (2002) Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators. Am J Physiology-Endocrinology Metabolism 282:E974\u0026ndash;E976\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmann T, Maegdefrau U, Hartmann A, Agaimy A, Marienhagen J, Weiss TS et al (2009) GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis. Am J Pathol 174:1544\u0026ndash;1552\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen X, Wang C, Liang N, Liu Z, Li X, Zhu ZJ et al (2021) Serum Metabolomics Identifies Dysregulated Pathways and Potential Metabolic Biomarkers for Hyperuricemia and Gout. Arthritis Rheumatol 73:1738\u0026ndash;1748\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarim S (2012) Hepatic expression and cellular distribution of the glucose transporter family. World J Gastroenterol ;18\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSzablewski L (2013) Expression of glucose transporters in cancers. Biochim et Biophys Acta (BBA) - Reviews Cancer 1835:164\u0026ndash;169\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdekola K, Rosen ST, Shanmugam M (2012) Glucose transporters in cancer metabolism. Curr Opin Oncol 24:650\u0026ndash;654\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMueckler M, Thorens B (2013) The SLC2 (GLUT) family of membrane transporters. Mol Aspects Med 34:121\u0026ndash;138\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiebeneicher H, Cleve A, Rehwinkel H, Neuhaus R, Heisler I, M\u0026uuml;ller T et al (2016) Identification and Optimization of the First Highly Selective GLUT1 Inhibitor BAY-876. ChemMedChem 11:2261\u0026ndash;2271\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang J, Zhao X, Guo Y, Liu Z, Wei S, Yuan Q et al (2022) Macrophage ALDH2 (Aldehyde Dehydrogenase 2) Stabilizing Rac2 Is Required for Efferocytosis Internalization and Reduction of Atherosclerosis Development. Arterioscler Thromb Vasc Biol 42:700\u0026ndash;716\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCilenti L, Di Gregorio J, Ambivero CT, Andl T, Liao R, Zervos AS (2020) Mitochondrial MUL1 E3 ubiquitin ligase regulates Hypoxia Inducible Factor (HIF-1α) and metabolic reprogramming by modulating the UBXN7 cofactor protein. Sci Rep ;10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen X, Cao M, Wang P, Chu S, Li M, Hou P et al (2022) The emerging roles of TRIM21 in coordinating cancer metabolism, immunity and cancer treatment. Front Immunol 13:968755\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSwatek KN, Komander D (2016) Ubiquitin modifications. Cell Res 26:399\u0026ndash;422\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer statistics, 2023. CA Cancer J Clin 73:17\u0026ndash;48\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHou G, Chen L, Liu G, Li L, Yang Y, Yan HX et al (2017) Aldehyde dehydrogenase-2 (ALDH2) opposes hepatocellular carcinoma progression by regulating AMP‐activated protein kinase signaling in mice. Hepatology 65:1628\u0026ndash;1644\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoroughs LK, DeBerardinis RJ (2015) Metabolic pathways promoting cancer cell survival and growth. Nat Cell Biol 17:351\u0026ndash;359\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWohlfart DP, Lou B, Middel CS, Morgenstern J, Fleming T, Sticht C et al (2022) Accumulation of acetaldehyde in aldh2.1(-/-) zebrafish causes increased retinal angiogenesis and impaired glucose metabolism. Redox Biol 50:102249\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa LL, Ding ZW, Yin PP, Wu J, Hu K, Sun AJ et al (2021) Hypertrophic preconditioning cardioprotection after myocardial ischaemia/reperfusion injury involves ALDH2-dependent metabolism modulation. Redox Biol 43:101960\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao R, Fang D, Wang J, Yu Y, Ye H, Kang P et al (2019) ALDH2 Overexpression Alleviates High Glucose-Induced Cardiotoxicity by Inhibiting NLRP3 Inflammasome Activation. J Diabetes Res 2019:4857921\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang C, Fan F, Cao Q, Shen C, Zhu H, Wang P et al (2016) Mitochondrial aldehyde dehydrogenase 2 deficiency aggravates energy metabolism disturbance and diastolic dysfunction in diabetic mice. J Mol Med (Berl) 94:1229\u0026ndash;1240\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmann T, Hellerbrand C (2009) GLUT1 as a therapeutic target in hepatocellular carcinoma. Expert Opin Ther Targets 13:1411\u0026ndash;1427\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOsthus RC, Shim H, Kim S, Li Q, Reddy R, Mukherjee M et al (2000) Deregulation of glucose transporter 1 and glycolytic gene expression by c-Myc. J Biol Chem 275:21797\u0026ndash;21800\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang C, Liu Z, Wang F, Zhang B, Zhang X, Guo P et al (2023) Nanomicelles for GLUT1-targeting hepatocellular carcinoma therapy based on NADPH depletion. Drug Deliv 30:2162160\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCohen NR, Knecht DA, Lodish HF (1996) Functional expression of rat GLUT 1 glucose transporter in Dictyostelium discoideum. Biochem J 315:971\u0026ndash;975\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee EE, Ma J, Sacharidou A, Mi W, Salato VK, Nguyen N et al (2015) A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome. Mol Cell 58:845\u0026ndash;853\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng N, Shabek N (2017) Ubiquitin Ligases: Structure, Function, and Regulation. Annu Rev Biochem 86:129\u0026ndash;157\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOzato K, Shin DM, Chang TH, Morse HC (2008) 3rd. TRIM family proteins and their emerging roles in innate immunity. Nat Rev Immunol 8:849\u0026ndash;860\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGu L, Zhu Y, Lin X, Tan X, Lu B, Li Y (2020) Stabilization of FASN by ACAT1-mediated GNPAT acetylation promotes lipid metabolism and hepatocarcinogenesis. Oncogene 39:2437\u0026ndash;2449\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng J, Huang Y, Zhang X, Yu Y, Wu S, Jiao J et al (2020) TRIM21 and PHLDA3 negatively regulate the crosstalk between the PI3K/AKT pathway and PPP metabolism. Nat Commun 11:1880\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee JH, Liu R, Li J, Zhang C, Wang Y, Cai Q et al (2017) Stabilization of phosphofructokinase 1 platelet isoform by AKT promotes tumorigenesis. Nat Commun 8:949\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGu M, Tan M, Zhou L, Sun X, Lu Q, Wang M et al (2022) Protein phosphatase 2Acα modulates fatty acid oxidation and glycolysis to determine tubular cell fate and kidney injury. Kidney Int 102:321\u0026ndash;336\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun A, Ren J (2013) ALDH2, a novel protector against stroke? Cell Res 23:874\u0026ndash;875\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa H, Guo R, Yu L, Zhang Y, Ren J (2011) Aldehyde dehydrogenase 2 (ALDH2) rescues myocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic aldehyde. Eur Heart J 32:1025\u0026ndash;1038\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi M, He X, Guo W, Yu H, Zhang S, Wang N et al (2020) Aldolase B suppresses hepatocellular carcinogenesis by inhibiting G6PD and pentose phosphate pathways. Nat Cancer 1:735\u0026ndash;747\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang N, Liu H, Liu G, Li M, He X, Yin C et al (2020) Yeast beta-D-glucan exerts antitumour activity in liver cancer through impairing autophagy and lysosomal function, promoting reactive oxygen species production and apoptosis. Redox Biol 32:101495\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu G, Shi A, Wang N, Li M, He X, Yin C et al (2020) Polyphenolic Proanthocyanidin-B2 suppresses proliferation of liver cancer cells and hepatocellular carcinogenesis through directly binding and inhibiting AKT activity. Redox Biol 37:101701\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa L, Tao Y, Duran A, Llado V, Galvez A, Barger JF et al (2013) Control of nutrient stress-induced metabolic reprogramming by PKCzeta in tumorigenesis. Cell 152:599\u0026ndash;611\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePortnoy VA, Scott DA, Lewis NE, Tarasova Y, Osterman AL, Palsson BO (2010) Deletion of genes encoding cytochrome oxidases and quinol monooxygenase blocks the aerobic-anaerobic shift in Escherichia coli K-12 MG1655. Appl Environ Microbiol 76:6529\u0026ndash;6540\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7102103/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7102103/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Aldehyde dehydrogenase 2 (ALDH2) detoxifies alcohol-derived acetaldehyde and lipid aldehydes from lipid peroxidation. A single nucleotide polymorphism of ALDH2 rs671, representing 30-50% East Asians and featuring ALDH2 deficiency, is associated with increased risk of hepatocellular carcinoma (HCC), but the underlying mechanism remains unclear. Here we demonstrate a non-catalytic role of ALDH2 in regulating glucose metabolism through post-translational control of GLUT1 stability. Using ALDH2 knockout and rs671 knock-in mice, we show that ALDH2 interacts with GLUT1 and promotes its K48-linked ubiquitination at lysine 256 via recruitment of E3 ligase TRIM21. ALDH2 deficiency reduces GLUT1 ubiquitination, stabilizes GLUT1 protein, enhances glycolytic flux, and promotes HCC development. These effects occur independently of alcohol exposure. Pharmacological inhibition or genetic silencing of GLUT1 reverses the tumor-promoting effect of ALDH2 deficiency in both xenograft and DEN-induced models. These findings reveal an unrecognized metabolic function of ALDH2 and nominate GLUT1 as a tractable vulnerability in ALDH2-deficient HCC.","manuscriptTitle":"ALDH2 Suppresses Glycolysis in Hepatocellular Carcinoma via TRIM21-Mediated GLUT1 Degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 05:31:46","doi":"10.21203/rs.3.rs-7102103/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6511b2c9-a47f-4814-a089-48a50959fbb7","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52848241,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Liver cancer/Hepatocellular carcinoma"},{"id":52848242,"name":"Biological sciences/Cell biology/Post-translational modifications/Ubiquitylation"}],"tags":[],"updatedAt":"2026-03-23T05:30:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-11 05:31:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7102103","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7102103","identity":"rs-7102103","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}