ER-associated degradation ligase HRD1 links ER stress to DNA damage repair by modulating the activity of DNA-PKcs.

Proteostasis and genomic integrity are respectively regulated by the endoplasmic reticulum-associated protein degradation (ERAD) and DNA damage repair signaling pathways, with both pathways essential for carcinogenesis and drug resistance. How these signaling pathways coordinate with each other remains unexplored. We found that ER stress specifically induces the DNA-PKcs-regulated nonhomologous end joining (NHEJ) pathway to amend DNA damage and impede cell death. Intriguingly, sustained ER stress rapidly decreased the activity of DNA-PKcs and DNA damage accumulated, facilitating a switch from adaptation to cell death. This DNA-PKcs inactivation was caused by increased KU70/KU80 protein degradation. Unexpectedly, the ERAD ligase HRD1 was found to efficiently destabilize the classic nuclear protein HDAC1 in the cytoplasm, by catalyzing HDAC1's polyubiquitination at lysine 74, at a late stage of ER stress. By abolishing HDAC1-mediated KU70/KU80 deacetylation, HRD1 transmits ER signals to the nucleus. The resulting enhanced KU70/KU80 acetylation provides binding sites for the nuclear E3 ligase TRIM25, resulting in the promotion of polyubiquitination and the degradation of KU70/KU80 proteins. Both in vitro and in vivo cancer models showed that genetic or pharmacological inhibition of HADC1 or DNA-PKcs sensitizes colon cancer cells to ER stress inducers, including the Food and Drug Administration-approved drug celecoxib. The antitumor effects of the combined approach were also observed in patient-derived xenograft models. These findings identify a mechanistic link between ER stress (ERAD) in the cytoplasm and DNA damage (NHEJ) pathways in the nucleus, indicating that combined anticancer strategies may be developed that induce severe ER stress while simultaneously inhibiting KU70/KU80/DNA-PKcs-mediated NHEJ signaling.

Excessive STAU1 condensate drives mTOR translation and autophagy dysfunction in neurodegeneration.

The double-stranded RNA-binding protein Staufen1 (STAU1) regulates a variety of physiological and pathological events via mediating RNA metabolism. STAU1 overabundance was observed in tissues from mouse models and fibroblasts from patients with neurodegenerative diseases, accompanied by enhanced mTOR signaling and impaired autophagic flux, while the underlying mechanism remains elusive. Here, we find that endogenous STAU1 forms dynamic cytoplasmic condensate in normal and tumor cell lines, as well as in mouse Huntington's disease knockin striatal cells. STAU1 condensate recruits target mRNA MTOR at its 5'UTR and promotes its translation both in vitro and in vivo, and thus enhanced formation of STAU1 condensate leads to mTOR hyperactivation and autophagy-lysosome dysfunction. Interference of STAU1 condensate normalizes mTOR levels, ameliorates autophagy-lysosome function, and reduces aggregation of pathological proteins in cellular models of neurodegenerative diseases. These findings highlight the importance of balanced phase separation in physiological processes, suggesting that modulating STAU1 condensate may be a strategy to mitigate the progression of neurodegenerative diseases with STAU1 overabundance.

ABHD7-mediated depalmitoylation of lamin A promotes myoblast differentiation.

LMNA gene mutation can cause muscular dystrophy, and post-translational modification plays a critical role in regulating its function. Here, we identify that lamin A is palmitoylated at cysteine 522, 588, and 591 residues, which are reversely catalyzed by palmitoyltransferase zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5) and depalmitoylase α/ß hydrolase domain 7 (ABHD7). Furthermore, the metabolite lactate promotes palmitoylation of lamin A by inhibiting the interaction between it and ABHD7. Interestingly, low-level palmitoylation of lamin A promotes, whereas high-level palmitoylation of lamin A inhibits, murine myoblast differentiation. Together, these observations suggest that ABHD7-mediated depalmitoylation of lamin A controls myoblast differentiation.

TET2 is required to suppress mTORC1 signaling through urea cycle with therapeutic potential.

Tumor development, involving both cell growth (mass accumulation) and cell proliferation, is a complex process governed by the interplay of multiple signaling pathways. TET2 mainly functions as a DNA dioxygenase, which modulates gene expression and biological functions via oxidation of 5mC in DNA, yet whether it plays a role in regulating cell growth remains unknown. Here we show that TET2 suppresses mTORC1 signaling, a major growth controller, to inhibit cell growth and promote autophagy. Mechanistically, TET2 functions as a 5mC "eraser" by mRNA oxidation, abolishes YBX1-HuR binding and promotes decay of urea cycle enzyme mRNAs, thus negatively regulating urea cycle and arginine production, which suppresses mTORC1 signaling. Therefore, TET2-deficient tumor cells are more sensitive to mTORC1 inhibition. Our results uncover a novel function for TET2 in suppressing mTORC1 signaling and inhibiting cell growth, linking TET2-mediated mRNA oxidation to cell metabolism and cell growth control. These findings demonstrate the potential of mTORC1 inhibition as a possible treatment for TET2-deficient tumors.

Feedback regulation of ubiquitination and phase separation of HECT E3 ligases.

Lipid homeostasis is essential for normal cellular functions and dysregulation of lipid metabolism is highly correlated with human diseases including neurodegenerative diseases. In the ubiquitin-dependent autophagic degradation pathway, Troyer syndrome-related protein Spartin activates and recruits HECT-type E3 Itch to lipid droplets (LDs) to regulate their turnover. In this study, we find that Spartin promotes the formation of Itch condensates independent of LDs. Spartin activates Itch through its multiple PPAY-motif platform generated by self-oligomerization, which targets the WW12 domains of Itch and releases the autoinhibition of the ligase. Spartin-induced activation and subsequent autoubiquitination of Itch lead to liquid-liquid phase separation (LLPS) of the poly-, but not oligo-, ubiquitinated Itch together with Spartin and E2 both in vitro and in living cells. LLPS-mediated condensation of the reaction components further accelerates the generation of polyubiquitin chains, thus forming a positive feedback loop. Such Itch-Spartin condensates actively promote the autophagy-dependent turnover of LDs. Moreover, we show that the catalytic HECT domain of Itch is sufficient to interact and phase separate with poly-, but not oligo-ubiquitin chains. HECT domains from other HECT E3 ligases also exhibit LLPS-mediated the promotion of ligase activity. Therefore, LLPS and ubiquitination are mutually interdependent and LLPS promotes the ligase activity of the HECT family E3 ligases.

Aldolase A Accelerates Cancer Progression by Modulating mRNA Translation and Protein Biosynthesis via Noncanonical Mechanisms.

Aldolase A (ALDOA), a crucial glycolytic enzyme, is often aberrantly expressed in various types of cancer. Although ALDOA has been reported to play additional roles beyond its conventional enzymatic role, its nonmetabolic function and underlying mechanism in cancer progression remain elusive. Here, it is shown that ALDOA promotes liver cancer growth and metastasis by accelerating mRNA translation independent of its catalytic activity. Mechanistically, ALDOA interacted with insulin- like growth factor 2 mRNA-binding protein 1 (IGF2BP1) to facilitate its binding to m6 A-modified eIF4G mRNA, thereby increasing eIF4G protein levels and subsequently enhancing overall protein biosynthesis in cells. Importantly, administration of GalNAc-conjugated siRNA targeting ALDOA effectively slows the tumor growth of orthotopic xenografts. Collectively, these findings uncover a previously unappreciated nonmetabolic function of ALDOA in modulating mRNA translation and highlight the potential of specifically targeting ALDOA as a prospective therapeutic strategy in liver cancer.

Increased α-HB links colorectal cancer and diabetes by potentiating NF-κB signaling.

Sufficient evidence has linked many different types of cancers and T2D through shared risk factors; however, the underlying mechanisms are not fully understood. α-Hydroxybutyrate (α-HB), a byproduct metabolite increased in diabetes and cancer, including colorectal cancer (CRC), triggers lactate dehydrogenase A (LDHA) nuclear translocation. Nuclear LDHA markedly extends NF-κB nuclear retention by interacting with phosphorylated p65, leading to an increase in TNF-α production, impaired insulin secretion and the exacerbation of azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC and high-fat diet (HFD)-induced type 2 diabetes. Furthermore, metformin interrupted this process by inhibiting the transcription of FOXM1 and c-MYC, the resultant downregulation of LDHA expression and α-HB-induced LDHA nuclear translocation. Thus, the results reveal the elevated α-HB level could be a novel shared risk factor of linking CRC, diabetes and the use of metformin treatment, as well as highlight the importance of preventing NF-κB activation for protecting against cancer and diabetes.

Enhanced BCAT1 activity and BCAA metabolism promotes RhoC activity in cancer progression.

Increased expression of branched-chain amino acid transaminase 1 or 2 (BCAT1 and BCAT2) has been associated with aggressive phenotypes of different cancers. Here we identify a gain of function of BCAT1 glutamic acid to alanine mutation at codon 61 (BCAT1E61A) enriched around 2.8% in clinical gastric cancer samples. We found that BCAT1E61A confers higher enzymatic activity to boost branched-chain amino acid (BCAA) catabolism, accelerate cell growth and motility and contribute to tumor development. BCAT1 directly interacts with RhoC, leading to elevation of RhoC activity. Notably, the BCAA-derived metabolite, branched-chain α-keto acid directly binds to the small GTPase protein RhoC and promotes its activity. BCAT1 knockout-suppressed cell motility could be rescued by expressing BCAT1E61A or adding branched-chain α-keto acid. We also identified that candesartan acts as an inhibitor of BCAT1E61A, thus repressing RhoC activity and cancer cell motility in vitro and preventing peritoneal metastasis in vivo. Our study reveals a link between BCAA metabolism and cell motility and proliferation through regulating RhoC activation, with potential therapeutic implications for cancers.

A metabolic clue for STING suppression.

A recent report by Heath et al. reveals that obesity could impair cancer immunogenicity and foster a type I interferon (IFN-I)-deprived tumor microenvironment through saturated fatty acid-mediated stimulator of interferon genes (STING) inhibition.

S-adenosyl-L-methionine supplementation alleviates damaged intestinal epithelium and inflammatory infiltration caused by Mat2a deficiency.

Methionine is important for intestinal development and homeostasis in various organisms. However, the underlying mechanisms are poorly understood. Here, we demonstrate that the methionine adenosyltransferase gene Mat2a is essential for intestinal development and that the metabolite S-adenosyl-L-methionine (SAM) plays an important role in intestinal homeostasis. Intestinal epithelial cell (IEC)-specific knockout of Mat2a exhibits impaired intestinal development and neonatal lethality. Mat2a deletion in the adult intestine reduces cell proliferation and triggers IEC apoptosis, leading to severe intestinal epithelial atrophy and intestinal inflammation. Mechanistically, we reveal that SAM maintains the integrity of differentiated epithelium and protects IECs from apoptosis by suppressing the expression of caspases 3 and 8 and their activation. SAM supplementation improves the defective intestinal epithelium and reduces inflammatory infiltration sequentially. In conclusion, our study demonstrates that methionine metabolism and its intermediate metabolite SAM play essential roles in intestinal development and homeostasis in mice.

Diet high in branched-chain amino acid promotes PDAC development by USP1-mediated BCAT2 stabilization.

BCAT2-mediated branched-chain amino acid (BCAA) catabolism is critical for pancreatic ductal adenocarcinoma (PDAC) development, especially at an early stage. However, whether a high-BCAA diet promotes PDAC development in vivo, and the underlying mechanism of BCAT2 upregulation, remain undefined. Here, we find that a high-BCAA diet promotes pancreatic intraepithelial neoplasia (PanIN) progression in LSL-KrasG12D/+ ; Pdx1-Cre (KC) mice. Moreover, we screened with an available deubiquitylase library which contains 31 members of USP family and identified that USP1 deubiquitylates BCAT2 at the K229 site. Furthermore, BCAA increases USP1 protein at the translational level via the GCN2-eIF2α pathway both in vitro and in vivo. More importantly, USP1 inhibition recedes cell proliferation and clone formation in PDAC cells and attenuates pancreas tumor growth in an orthotopic transplanted mice model. Consistently, a positive correlation between USP1 and BCAT2 is found in KC; LSL-KrasG12D/+ ; p53flox/+ ; Pdx1-Cre mice and clinical samples. Thus, a therapeutic targeting USP1-BCAT2-BCAA metabolic axis could be considered as a rational strategy for treatment of PDAC and precisive dietary intervention of BCAA has potentially translational significance.

Intracellular complement C5a/C5aR1 stabilizes ß-catenin to promote colorectal tumorigenesis.

Complement is operative in not only the extracellular but also the intracellular milieu. However, little is known about the role of complement activation inside tumor cells. Here, we report that intracellular C5 is cleaved by cathepsin D (CTSD) to produce C5a in lysosomes and endosomes of colonic cancer cells. After stimulation by C5a, intracellular C5aR1 assembles a complex with KCTD5/cullin3/Roc-1 and ß-catenin to promote the switch of polyubiquitination of ß-catenin from K48 to K63, which enhances ß-catenin stability. Genetic loss or pharmacological blockade of C5aR1 dramatically impedes colorectal tumorigenesis at least by destabilizing ß-catenin. In human colorectal cancer specimens, high levels of C5aR1, C5a, and CTSD are closely correlated with elevated ß-catenin levels and a poor prognosis. Importantly, intracellular C5a/C5aR1-mediated ß-catenin stabilization is also observed ubiquitously in other cell types. Collectively, we identify a machinery for ß-catenin activation and provide a potential target for tumor prevention and treatment.

Dietary folate drives methionine metabolism to promote cancer development by stabilizing MAT IIA.

Folic acid, served as dietary supplement, is closely linked to one-carbon metabolism and methionine metabolism. Previous clinical evidence indicated that folic acid supplementation displays dual effect on cancer development, promoting or suppressing tumor formation and progression. However, the underlying mechanism remains to be uncovered. Here, we report that high-folate diet significantly promotes cancer development in mice with hepatocellular carcinoma (HCC) induced by DEN/high-fat diet (HFD), simultaneously with increased expression of methionine adenosyltransferase 2A (gene name, MAT2A; protein name, MATIIα), the key enzyme in methionine metabolism, and acceleration of methionine cycle in cancer tissues. In contrast, folate-free diet reduces MATIIα expression and impedes HFD-induced HCC development. Notably, methionine metabolism is dynamically reprogrammed with valosin-containing protein p97/p47 complex-interacting protein (VCIP135) which functions as a deubiquitylating enzyme to bind and stabilize MATIIα in response to folic acid signal. Consistently, upregulation of MATIIα expression is positively correlated with increased VCIP135 protein level in human HCC tissues compared to adjacent tissues. Furthermore, liver-specific knockout of Mat2a remarkably abolishes the advocating effect of folic acid on HFD-induced HCC, demonstrating that the effect of high or free folate-diet on HFD-induced HCC relies on Mat2a. Moreover, folate and multiple intermediate metabolites in one-carbon metabolism are significantly decreased in vivo and in vitro upon Mat2a deletion. Together, folate promotes the integration of methionine and one-carbon metabolism, contributing to HCC development via hijacking MATIIα metabolic pathway. This study provides insight into folate-promoted cancer development, strongly recommending the tailor-made folate supplement guideline for both sub-healthy populations and patients with cancer expressing high level of MATIIα expression.

CRISPR/Cas9 Screens Reveal that Hexokinase 2 Enhances Cancer Stemness and Tumorigenicity by Activating the ACSL4-Fatty Acid ß-Oxidation Pathway.

Metabolic reprogramming is often observed in carcinogenesis, but little is known about the aberrant metabolic genes involved in the tumorigenicity and maintenance of stemness in cancer cells. Sixty-seven oncogenic metabolism-related genes in liver cancer by in vivo CRISPR/Cas9 screening are identified. Among them, acetyl-CoA carboxylase 1 (ACC1), aldolase fructose-bisphosphate A (ALDOA), fatty acid binding protein 5 (FABP5), and hexokinase 2 (HK2) are strongly associated with stem cell properties. HK2 further facilitates the maintenance and self-renewal of liver cancer stem cells. Moreover, HK2 enhances the accumulation of acetyl-CoA and epigenetically activates the transcription of acyl-CoA synthetase long-chain family member 4 (ACSL4), leading to an increase in fatty acid ß-oxidation activity. Blocking HK2 or ACSL4 effectively inhibits liver cancer growth, and GalNac-siHK2 administration specifically targets the growth of orthotopic tumor xenografts. These results suggest a promising therapeutic strategy for the treatment of liver cancer.

Palmitoylation of MDH2 by ZDHHC18 activates mitochondrial respiration and accelerates ovarian cancer growth.

Epithelial ovarian cancer (EOC) exhibits strong dependency on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to fuel anabolic process. Here, we show that malate dehydrogenase 2 (MDH2), a key enzyme of the TCA cycle, is palmitoylated at cysteine 138 (C138) residue, resulting in increased activity of MDH2. We next identify that ZDHHC18 acts as a palmitoyltransferase of MDH2. Glutamine deprivation enhances MDH2 palmitoylation by increasing the binding between ZDHHC18 and MDH2. MDH2 silencing represses mitochondrial respiration as well as ovarian cancer cell proliferation both in vitro and in vivo. Intriguingly, re-expression of wild-type MDH2, but not its palmitoylation-deficient C138S mutant, sustains mitochondrial respiration and restores the growth as well as clonogenic capability of ovarian cancer cells. Notably, MDH2 palmitoylation level is elevated in clinical cancer samples from patients with high-grade serous ovarian cancer. These observations suggest that MDH2 palmitoylation catalyzed by ZDHHC18 sustains mitochondrial respiration and promotes the malignancy of ovarian cancer, yielding possibilities of targeting ZDHHC18-mediated MDH2 palmitoylation in the treatment of EOC.

A pathway-guided strategy identifies a metabolic signature for prognosis prediction and precision therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly lethal and heterogeneous disease with a poor prognosis and no effective treatments. Herein, we presented a pathway-guided computational framework to establish a metabolic signature with the capacity for HCC prognosis prediction. By using the TCGA dataset as a training cohort (n = 365), we built an eight-gene (ACADS, ALDH1A2, FTCD, GOT2, GPX7, HADHA, LDHA and UGT2A1) risk score called the MGP score from the 20 metabolic pathways downregulated in HCC. The robustness of the MGP model was successfully validated in seven other independent cohorts (LIRI-JP, n = 231; Chinese, n = 159; GSE148355, n = 33; GSE14520, n = 225; GSE54236, n = 81; E-TABM-36, n = 41; and qPCR, n = 126). Moreover, three subtypes, L, H1 and H2, with distinct clinical outcomes were further stratified by using 761 HCC patients in the combined RNA-Seq cohort. Further analysis identified strong negative associations between metabolic pathways and other molecular features, including immune infiltration, expression of immune checkpoint genes, and hypoxic conditions, among the three subtypes. In 81 liver cancer cell lines, the MGP score indicated sensitivity to three preclinical agents (erastin, piperlongumine and PI-103), which may have potential therapeutic implications for the high-MGP score subtypes H1 and H2. Overall, our analysis highlights the potential of applying the MGP score for prognosis prediction and precision therapy for HCC.